Anti-tau antibodies and methods of use

ABSTRACT

The invention provides anti-Tau antibodies and methods of using the same.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No. 16/893,678, filed Jun. 5, 2020, which is a continuation of U.S. patent application Ser. No. 16/420,673, filed May 23, 2019, which is a continuation of International Application No. PCT/US2017/064869, filed Dec. 6, 2017, which claims the benefit of priority of U.S. Provisional Application No. 62/431,180, filed Dec. 7, 2016, each of which is incorporated by reference herein in its entirety for any purpose.

FIELD OF THE INVENTION

The present invention relates to anti-Tau antibodies and methods of using the same.

BACKGROUND

Neurofibrillary tangles and neuropil threads (NTs) are the major neuropathological hallmarks of Alzheimer's Disease (AD). NTs are composed of the microtubule-associated Tau protein that has undergone post-translational modifications including phosphorylation, and develop by aggregation of hyperphosphorylated Tau conformers. AD shares this pathology with many neurodegenerative tauopathies, in particularly with certain types of frontotemporal dementia (FTD). Tau protein appears to be a major player in the cognitive demise in AD and related neurodegenerative tauopathies.

Therapeutic approaches that target Tau protein are scarce and comprise mainly inhibitors of the kinases that are thought to increase the phosphorylation of Tau to pathological levels, and compounds that block the cytoplasmic aggregation of hyper-phosphorylated Tau protein. These approaches suffer various draw-backs of specificity and efficacy. There is a need for additional therapeutic agents that target the pathological protein conformers that are known or presumed to cause neurodegenerative disorders.

SUMMARY

The invention provides anti-Tau antibodies and methods of using the same. In some embodiments, antibodies with high affinity for human and cynomolgus monkey Tau are provided. In some embodiments, the antibodies have an affinity for human Tau of less than 1 nM or less than 0.5 nM or less than 0.3 nM as measured, for example, by surface plasmon resonance.

In some embodiments, an isolated antibody that binds to human Tau is provided, wherein the antibody binds to monomeric Tau, oligomeric Tau, non-phosphorylated Tau, and phosphorylated Tau. In some embodiments, the antibody binds an epitope within amino acids 2 to 24 of mature human Tau. In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the antibody is a human, humanized, or chimeric antibody. In some embodiments, the antibody is an antibody fragment that binds human Tau. In some embodiments, the human Tau comprises the sequence of SEQ ID NO: 2.

In some embodiments, the antibody comprises HVR-H1 comprising an amino acid sequence of SEQ ID NO: 605; HVR-H2 comprising an amino acid sequence of SEQ ID NO: 606; and HVR-H3 comprising an amino acid sequence of SEQ ID NO: 607.

In some embodiments, the antibody comprises HVR-L1 comprising an amino acid sequence of SEQ ID NO: 608; HVR-L2 comprising an amino acid sequence of SEQ ID NO: 609; and HVR-L3 comprising an amino acid sequence of SEQ ID NO: 610.

In some embodiments, the antibody comprises HVR-H1 comprising an amino acid sequence of SEQ ID NO: 605; HVR-H2 comprising an amino acid sequence of SEQ ID NO: 606; HVR-H3 comprising an amino acid sequence of SEQ ID NO: 607; HVR-L1 comprising an amino acid sequence of SEQ ID NO: 608; HVR-L2 comprising an amino acid sequence of SEQ ID NO: 609; and HVR-L3 comprising an amino acid sequence of SEQ ID NO: 610.

In some embodiments, the antibody comprises:

-   -   a) a heavy chain variable region (VH) comprising a sequence that         is at least 95% identical to SEQ ID NO: 603;     -   b) a light chain variable region (VL) comprising a sequence that         is at least 95% identical to SEQ ID NO: 604;     -   c) a VH as in (a) and a VL as in (b);     -   d) a heavy chain variable region (VH) comprising a sequence that         is at least 95% identical to SEQ ID NO: 614;     -   e) a light chain variable region (VL) comprising a sequence that         is at least 95% identical to SEQ ID NO: 615;     -   f) a VH as in (d) and a VL as in (e);     -   g) a heavy chain variable region (VH) comprising a sequence that         is at least 95% identical to SEQ ID NO: 619;     -   h) a light chain variable region (VL) comprising a sequence that         is at least 95% identical to SEQ ID NO: 620;     -   i) a VH as in (g) and a VL as in (h).

In some embodiments, the antibody comprises:

-   -   a) a heavy chain variable region (VH) comprising SEQ ID NO: 603;     -   b) a light chain variable region (VL) comprising SEQ ID NO: 604;     -   c) a VH as in (a) and a VL as in (b);     -   d) a heavy chain variable region (VH) comprising the sequence of         SEQ ID NO: 614;     -   e) a light chain variable region (VL) comprising the sequence of         SEQ ID NO: 615;     -   f) a VH as in (d) and a VL as in (e);     -   g) a heavy chain variable region (VH) comprising the sequence of         SEQ ID NO: 619;     -   h) a light chain variable region (VL) comprising the sequence of         SEQ ID NO: 620;     -   i) a VH as in (g) and a VL as in (h).

In some embodiments, the antibody comprises a heavy chain variable region comprising a sequence selected from SEQ ID NOs: 603, 614, and 619; and a light chain variable region comprising a sequence selected from SEQ ID NOs: 604, 615, and 620. In some embodiments, the antibody comprises a heavy chain variable region comprising a sequence selected from SEQ ID NOs: 340, 603, 614, and 619; and a light chain variable region comprising a sequence selected from SEQ ID NOs: 604, 615, and 620. In some embodiments, the antibody comprises a heavy chain variable region comprising a sequence selected from SEQ ID NOs: 603, 614, and 619; and a light chain variable region comprising a sequence selected from SEQ ID NOs: 341, 604, 615, and 620.

In some embodiments, the antibody comprises (a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 603 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 604; (b) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 614 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 615; or (c) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 619 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 620.

In some embodiments, the antibody comprises (a) a heavy chain comprising the amino acid sequence of SEQ ID NO: 611 or SEQ ID NO: 612 and a light chain comprising the amino acid sequence of SEQ ID NO: 613; (b) a heavy chain comprising the amino acid sequence of SEQ ID NO: 616 or SEQ ID NO: 617 and a light chain comprising the amino acid sequence of SEQ ID NO: 618; or (c) a heavy chain comprising the amino acid sequence of SEQ ID NO: 621 or SEQ ID NO: 622 and a light chain comprising the amino acid sequence of SEQ ID NO: 623.

In some embodiments, an isolated antibody that binds to human Tau is provided, wherein the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 611 or SEQ ID NO: 612 and a light chain comprising the amino acid sequence of SEQ ID NO: 613. In some embodiments, an isolated antibody that binds to human Tau is provided, wherein the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 611 and a light chain comprising the amino acid sequence of SEQ ID NO: 613. In some embodiments, an isolated antibody that binds to human Tau is provided, wherein the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 612 and a light chain comprising the amino acid sequence of SEQ ID NO: 613. In some embodiments, an isolated antibody that binds to human Tau is provided, wherein the antibody comprises a heavy chain consisting of the amino acid sequence of SEQ ID NO: 611 or SEQ ID NO: 612 and a light chain consisting of the amino acid sequence of SEQ ID NO: 613. In some embodiments, an isolated antibody that binds to human Tau is provided, wherein the antibody comprises a heavy chain consisting of the amino acid sequence of SEQ ID NO: 611 and a light chain consisting of the amino acid sequence of SEQ ID NO: 613. In some embodiments, an isolated antibody that binds to human Tau is provided, wherein the antibody comprises a heavy chain consisting of the amino acid sequence of SEQ ID NO: 612 and a light chain consisting of the amino acid sequence of SEQ ID NO: 613.

In some embodiments, an isolated antibody that binds to human Tau is provided, wherein the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 616 or 617 and a light chain comprising the amino acid sequence of SEQ ID NO: 618. In some embodiments, an isolated antibody that binds to human Tau is provided, wherein the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 616 and a light chain comprising the amino acid sequence of SEQ ID NO: 618. In some embodiments, an isolated antibody that binds to human Tau is provided, wherein the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 617 and a light chain comprising the amino acid sequence of SEQ ID NO: 618. In some embodiments, an isolated antibody that binds to human Tau is provided, wherein the antibody comprises a heavy chain consisting of the amino acid sequence of SEQ ID NO: 616 or SEQ ID NO: 617 and a light chain consisting of the amino acid sequence of SEQ ID NO: 618. In some embodiments, an isolated antibody that binds to human Tau is provided, wherein the antibody comprises a heavy chain consisting of the amino acid sequence of SEQ ID NO: 616 and a light chain consisting of the amino acid sequence of SEQ ID NO: 618. In some embodiments, an isolated antibody that binds to human Tau is provided, wherein the antibody comprises a heavy chain consisting of the amino acid sequence of SEQ ID NO: 617 and a light chain consisting of the amino acid sequence of SEQ ID NO: 618.

In some embodiments, an isolated antibody that binds to human Tau is provided, wherein the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 621 or SEQ ID NO: 622 and a light chain comprising the amino acid sequence of SEQ ID NO: 623. In some embodiments, an isolated antibody that binds to human Tau is provided, wherein the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 621 and a light chain comprising the amino acid sequence of SEQ ID NO: 623. In some embodiments, an isolated antibody that binds to human Tau is provided, wherein the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 622 and a light chain comprising the amino acid sequence of SEQ ID NO: 623. In some embodiments, an isolated antibody that binds to human Tau is provided, wherein the antibody comprises a heavy chain consisting of the amino acid sequence of SEQ ID NO: 621 or SEQ ID NO: 622 and a light chain consisting of the amino acid sequence of SEQ ID NO: 623. In some embodiments, an isolated antibody that binds to human Tau is provided, wherein the antibody comprises a heavy chain consisting of the amino acid sequence of SEQ ID NO: 621 and a light chain consisting of the amino acid sequence of SEQ ID NO: 623. In some embodiments, an isolated antibody that binds to human Tau is provided, wherein the antibody comprises a heavy chain consisting of the amino acid sequence of SEQ ID NO: 622 and a light chain consisting of the amino acid sequence of SEQ ID NO: 623.

In any of the embodiments described herein, the antibody may be an IgG1 or an IgG4 antibody. In any of the embodiments described herein, the antibody may be an IgG4 antibody. In some such embodiments, the antibody comprises M252Y, S254T, and T256E mutations. In any of the embodiments described herein, the antibody may comprise an S228P mutation. In any of the embodiments described herein, the antibody may comprise S228P, M252Y, S254T, and T256E mutations. In any of the embodiments described herein, the antibody may be an IgG4 antibody comprising S228P, M252Y, S254T, and T256E mutations. In some embodiments, the antibody is an antibody fragment. In any of the embodiments described herein, the antibody may be an IgG4 antibody comprising S228P, M252Y, S254T, and T256E mutations, and lacking the C-terminal lysine (des-K) of the heavy chain constant region. The C-terminal lysine of the heavy chain constant region may be removed, for example, during purification of the antibody or by recombinant engineering of the nucleic acid encoding the antibody such that the C-terminal lysine is not encoded.

In some embodiments, an isolated antibody that binds human Tau is provided, wherein the antibody binds each of monomeric Tau, phosphorylated Tau, non-phosphorylated Tau, and oligomeric Tau with a K_(D) of less than 100 nM, less than 75 nM, less than 50 nM, less than 10 nM, less than 5 nM, or less than 1 nM. In some embodiments, the antibody binds cynomolgus monkey Tau. In some embodiments, the antibody binds to human monomeric Tau and/or cynomolgus monkey monomeric Tau with a K_(D) of less than 1 nM.

In some embodiments, an isolated nucleic acid is provided, wherein the isolated nucleic acid encodes an antibody described herein. In some embodiments, a host cell is provided, wherein the host cell comprises an isolated nucleic acid that encodes an antibody described herein. In some embodiments, a method of producing an antibody is provided, comprising culturing the host cell under conditions suitable for producing the antibody.

In some embodiments, an immunoconjugate is provided, wherein the immunoconjugate comprises an isolated antibody described herein and a therapeutic agent. In some embodiments, a labeled antibody is provided, comprising an antibody described herein and a detectable label.

In some embodiments, a pharmaceutical composition is provided, comprising an isolated antibody described herein and a pharmaceutically acceptable carrier.

In some embodiments, a method of treating a Tau protein associated disease is provided, comprising administering to an individual with a Tau protein related disease an antibody described herein or a pharmaceutical composition comprising an antibody described herein. In some embodiments, the Tau protein associated disease is a tauopathy. In some embodiments, the tauopathy is a neurodegenerative tauopathy. In some embodiments, the tauopathy is selected from Alzheimer's Disease, amyotrophic lateral sclerosis, Parkinson's disease, Creutzfeldt-Jacob disease, Dementia pugilistica, Down's Syndrome, GerstmannStraussler-Scheinker disease, inclusion-body myositis, prion protein cerebral amyloid angiopathy, traumatic brain injury, amyotrophic lateral sclerosis/parkinsonism-dementia complex of Guam, Non-Guamanian motor neuron disease with neurofibrillary tangles, argyrophilic grain dementia, corticobasal degeneration, diffuse neurofibrillary tangles with calcification, frontotetemporal dementia, frontotemporal dementia with parkinsonism linked to chromosome 17, Hallevorden-Spatz disease, multiple system atrophy, Niemann-Pick disease type C, Pallido-ponto-nigral degeneration, Pick's disease, progressive subcortical gliosis, progressive supranuclear palsy, Subacute sclerosing panencephalitis, Tangle only dementia, Postencephalitic Parkinsonism, and Myotonic dystrophy. In some embodiments, the tauopathy is Alzheimer's disease or progressive supranuclear palsy. In some embodiments, the Tau protein associated disease is selected from PART (primary age-related Tauopathy), tangle predominant dementia, subacute sclerosis panencephalopathy, chronic traumatic encephalopathy (CTE), white matter tauopathy with globular glial inclusions, Lewy body dementia (LBD), mild cognitive impairment (MCI), glaucoma, familial British dementia, familiar Danish dementia, Guadeloupean Parkinsonism, neurodegeneration with brain iron accumulation, SLC9A6-related mental retardation, multiple sclerosis, HIV-related dementia, senile cardiac amyloidosis, and Huntington's disease.

In some embodiments, a method of retaining or increasing cognitive memory capacity or slowing memory loss in an individual is provided, comprising administering an antibody described herein or a pharmaceutical composition comprising an antibody described herein.

In some embodiments, a method of reducing the level of Tau protein, non-phosphorylated Tau protein, phosphorylated Tau protein, or hyperphosphorylated Tau protein in an individual is provided, comprising administering an antibody described herein or a pharmaceutical composition comprising an antibody described herein.

In some embodiments, an isolated antibody described herein is provided for use as a medicament. In some embodiments, an isolated antibody described herein is provided for use in treating a tauopathy in an individual. In some embodiments, the tauopathy is a neurodegenerative tauopathy. In some embodiments, the tauopathy is selected from Alzheimer's Disease, amyotrophic lateral sclerosis, Parkinson's disease, Creutzfeldt-Jacob disease, Dementia pugilistica, Down's Syndrome, Gerstmann-Sträussler-Scheinker disease, inclusion-body myositis, prion protein cerebral amyloid angiopathy, traumatic brain injury, amyotrophic lateral sclerosis/parkinsonism-dementia complex of Guam, Non-Guamanian motor neuron disease with neurofibrillary tangles, argyrophilic grain dementia, corticobasal degeneration, diffuse neurofibrillary tangles with calcification, frontotetemporal dementia, frontotemporal dementia with parkinsonism linked to chromosome 17, Hallevorden-Spatz disease, multiple system atrophy, Niemann-Pick disease type C, Pallido-ponto-nigral degeneration, Pick's disease, progressive subcortical gliosis, progressive supranuclear palsy, Subacute sclerosing panencephalitis, Tangle only dementia, Postencephalitic Parkinsonism, and Myotonic dystrophy. In some embodiments, the tauopathy is Alzheimer's disease or progressive supranuclear palsy. In some embodiments, the Tau protein associated disease is selected from PART (primary age-related Tauopathy), tangle predominant dementia, subacute sclerosis panencephalopathy, chronic traumatic encephalopathy (CTE), white matter tauopathy with globular glial inclusions, Lewy body dementia (LBD), mild cognitive impairment (MCI), glaucoma, familial British dementia, familiar Danish dementia, Guadeloupean Parkinsonism, neurodegeneration with brain iron accumulation, SLC9A6-related mental retardation, multiple sclerosis, HIV-related dementia, senile cardiac amyloidosis, and Huntington's disease.

In some embodiments, an isolated antibody described herein is provided for use in retaining or increasing cognitive memory capacity or slowing memory loss in an individual. In some embodiments, an isolated antibody described herein is provided for use in reducing the level of Tau protein, phosphorylated Tau protein, non-phosphorylated Tau protein, or hyperphosphorylated Tau protein in an individual.

In some embodiments, use of an antibody described herein is provided for manufacture of a medicament for treating a Tau protein associated disease in an individual. In some embodiments, the Tau protein associate disease is a tauopathy. In some embodiments, the tauopathy is a neurodegenerative tauopathy. In some embodiments, the tauopathy is selected from Alzheimer's Disease, amyotrophic lateral sclerosis, Parkinson's disease, Creutzfeldt-Jacob disease, Dementia pugilistica, Down's Syndrome, Gerstmann-Straussler-Scheinker disease, inclusion-body myositis, prion protein cerebral amyloid angiopathy, traumatic brain injury, amyotrophic lateral sclerosis/parkinsonism-dementia complex of Guam, Non-Guamanian motor neuron disease with neurofibrillary tangles, argyrophilic grain dementia, corticobasal degeneration, diffuse neurofibrillary tangles with calcification, frontotetemporal dementia, frontotemporal dementia with parkinsonism linked to chromosome 17, Hallevorden-Spatz disease, multiple system atrophy, Niemann-Pick disease type C, Pallido-ponto-nigral degeneration, Pick's disease, progressive subcortical gliosis, progressive supranuclear palsy, Subacute sclerosing panencephalitis, Tangle only dementia, Postencephalitic Parkinsonism, and Myotonic dystrophy. In some embodiments, the tauopathy is Alzheimer's disease or progressive supranuclear palsy. In some embodiments, the Tau protein associated disease is selected from PART (primary age-related Tauopathy), tangle predominant dementia, subacute sclerosis panencephalopathy, chronic traumatic encephalopathy (CTE), white matter tauopathy with globular glial inclusions, Lewy body dementia (LBD), mild cognitive impairment (MCI), glaucoma, familial British dementia, familiar Danish dementia, Guadeloupean Parkinsonism, neurodegeneration with brain iron accumulation, SLC9A6-related mental retardation, multiple sclerosis, HIV-related dementia, senile cardiac amyloidosis, and Huntington's disease.

In some embodiments, use of an antibody described herein is provided for manufacture of a medicament for retaining or increasing cognitive memory capacity or slowing memory loss in an individual.

In some embodiments, a method of detecting neurofibrillary tangles, neuropil threads, or dystrophic neuritis is provided, comprising contacting a sample with an antibody described herein. In some embodiments, the sample is a brain sample, a cerebrospinal fluid sample, or a blood sample.

In any of the embodiments described herein, a method or use may comprise administering an antibody described herein in combination with at least one additional therapy. Non-limiting examples of additional therapies include neurological drugs, corticosteroids, antibiotics, antiviral agents, and other therapeutic agents. Such other therapeutic agents include, but are not limited to, other anti-Tau antibodies, antibodies against amyloid-beta, antibodies against beta-secretase 1 (“BACE1”), and inhibitors of beta-secretase 1.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A-F. Binding of antibodies to hyperphosphorylated Tau (pTau) was compared to non-phosphorylated Tau using an ELISA. Results are expressed in optical densities (O.D.).

FIG. 2A-E. Binding of antibodies to oligomeric Tau was assessed using an oligo- and monoTau capture ELISA. Results are expressed in optical densities (O.D.).

FIG. 3 . The three panTau antibodies tested show binding to soluble Tau in brain lysates from Alzheimer's disease (AD) and matched control donors using a Western blot (WB) assay. Protein extracts from AD and control brain lysates, and six isoforms of recombinant human Tau, were run on SDS-PAGE and membranes blotted with three panTau antibodies (37D3-H9, 94B2-C1, and 125B11-H3). Lanes with AD samples are labeled as AD18, AD24, and AD27, and lanes with control samples are labeled as C25 and C21. The lanes run with six isoforms of recombinant human Tau are labeled as hTau ladder.

FIG. 4A-C. PanTau antibodies show binding to soluble Tau in brain lysates from AD and matched control donors using a Tau capture ELISA. Data is shown for three panTau antibodies, 37D3-H9, 94B2-C1, and 125B11-H3. Results are expressed in optical densities (O.D.), with mean values±SD, N=2.

FIG. 5 . Sensorgrams showing 37D3-H9 binding as a Fab (left panel) and as an IgG (right panel) to human Tau monomer covalently coupled to a Biacore chip surface. A 1:1 binding model has been applied and is shown as an overlay. The x-axis indicates time (units=seconds). The y-axis indicated Resonance Units (RU).

FIG. 6 . Overlaid sensorgrams showing binding of hu37D3-H9.v5 samples t=0 (left panel) and t=2 weeks (right panel) to human Tau monomer at 3.1, 6.3, 12.5, 25, 25, 50 and 100 nM. A 1:1 binding model has been applied and is also shown in this figure. The x-axis indicates time (units=seconds). The y-axis indicated Resonance Units (RU).

FIG. 7 . Binding of hu37D3-H9.v5 and hu37D3-H9.v5 N28D to monomeric Tau individually (left panel shows hu37D3-H9.v5 and middle panel shows hu37D3-H9.v5 N28D) and mixed at a 1:1 ratio (right panel). The x-axis indicates time (units=seconds). The y-axis indicated Resonance Units (RU).

FIG. 8A-D. Affinity, stability index and sequences of the ninety 37D3-H9 variants screened for potential improved stability. For clarity, values obtained with an unstressed control antibody (hu37D3-H9.v5 hIgG1) run at the beginning, middle and end of each experiment are shown in both sections of the table.

FIG. 9 . Structural model of the 37D3-H9 Fv region showing the positions of residues 28 to 33 (NGNTYF motif) of the light chain and relative positions of residues 28 and 33. Note that residue 33, mutated in hu37D3.v28.A4 to Leu, is not nearby the unstable Asn-28 residue. The dotted line shows a hydrogen bond between residues Asn-28 and Tyr-32. Figure generated using MOE software package (Chemical Computing Group).

FIG. 10 shows pharmacokinetics of anti-Tau antibody 37D3-H9 in mice following a single 10 mg/kg intravenous or intraperitoneal injection.

FIG. 11 shows pharmacokinetics of hu37D3.v28.A4 hIgG4-S228P and hu37D3.v28.A4 hIgG4-S228P.YTE in cynomolgus monkeys following a single IV bolus injection at a dose of 1 mg/kg.

FIG. 12A-C. Binding of certain anti-Tau antibodies to Tau fragments. (A) Binding of certain anti-Tau antibodies to Tau fragments 1-15, 10-24, 19-33, 28-42, 37-51, and 46-60 is shown. (B) Binding of antibody 37D3-H9 mIgG2a to Tau fragments 10-44, 10-24, 2-24, 2-34, and full-length Tau. (C) Binding of antibody hu37D3-H9.v5 hIgG1 to Tau fragments 10-44, 10-24, 2-24, 2-34, and full-length Tau.

FIG. 13A-B. Effect of effector function on Tau toxicity in neuron-microglia co-cultures. (A) Percent MAP2 fragmentation in co-cultures contacted with various antibodies and oligomeric Tau. (B) Images of neurons (top panels) and neuron-microglia co-cultures (bottom panels) contacted with various antibodies and oligomeric Tau.

FIG. 14 . pTau212/214 levels in the hippocampus of mice administered anti-tau 37D3-H9 WT IgG2a or anti-tau 37D3-H9 DANG IgG2.

FIG. 15 . Comparison of human and cynomolgus monkey Tau sequences. The epitope for antibody 37D3-H9 is indicated.

FIG. 16 shows pharmacokinetics of anti-Tau antibody 94B2-C1 in mice following a single 10 mg/kg intravenous or intraperitoneal injection.

FIG. 17 shows pharmacokinetics of anti-Tau antibody 125B11-H3 in mice following a single 10 mg/kg intravenous or intraperitoneal injection.

FIG. 18 shows an alignment of the kappa 1 light chain variable regions of hu37D3-H9.v1, hu37D3-H9.v39, hu37D3-H9.v40, and hu37D3-H9.v41.

FIG. 19A-B show plasma antibody concentration (A) and CSF antibody concentration (B) in cynomolgus monkeys following a single IV injection of the indicated antibody at 50 mg/kg.

FIG. 20 shows plasma total Tau concentration and plasma antibody concentration in cynomolgus monkeys following a single IV injection of the indicated antibody at 50 mg/kg.

FIGS. 21A-D show antibody concentration in various regions of cynomolgus monkey brain 2 days and 10 days following a single IV injection of hu37D3.v28.A4 hIgG4-S228P (A) and hu37D3.v28.A4 hIgG4-S228P.YTE (B) at 50 mg/kg; average antibody concentration in brain (C); % brain:plasma antibody concentration (D).

FIGS. 22A-B show the concentration of antibody in cynomolgus monkey brain at various time points following a single IV injection of the indicated antibody at 50 mg/kg, plotted in logarithmic (A) and linear (B) scale.

FIGS. 23A-E show the concentration of antibody in the hippocampus (A), cerebellum (B), frontal cortex (C), CSF (D), and plasma (E) of cynomolgus monkeys at various time points following a single IV injection of the indicated antibody at 50 mg/kg.

FIGS. 24A-B show average (A) and individual (B) plasma total Tau concentration over time in cynomolgus monkeys following a single IV injection of the indicated antibody at 50 mg/kg.

FIG. 25A-B shows sequence alignment between the affinity-matured hu37D3-H9.v76, hu37D3-H9.v83, and hu37D3-H9.v93 antibodies versus the parent hu37D3-H9.v28.A4 antibody. Amino acid differences are shaded in black.

FIGS. 26A-B show affinity measurements of the hu37D3-H9.v76 for human tau monomer (A) and cynomolgus monkey tau monomer (B).

DETAILED DESCRIPTION OF EMBODIMENTS OF THE INVENTION I. Definitions

An “acceptor human framework” for the purposes herein is a framework comprising the amino acid sequence of a light chain variable domain (VL) framework or a heavy chain variable domain (VH) framework derived from a human immunoglobulin framework or a human consensus framework, as defined below. An acceptor human framework “derived from” a human immunoglobulin framework or a human consensus framework may comprise the same amino acid sequence thereof, or it may contain amino acid sequence changes. In some embodiments, the number of amino acid changes are 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less. In some embodiments, the VL acceptor human framework is identical in sequence to the VL human immunoglobulin framework sequence or human consensus framework sequence.

“Affinity” refers to the strength of the sum total of noncovalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless indicated otherwise, as used herein, “binding affinity” refers to intrinsic binding affinity which reflects a 1:1 interaction between members of a binding pair (e.g., antibody and antigen). The affinity of a molecule X for its partner Y can generally be represented by the dissociation constant (K_(D)). Affinity can be measured by common methods known in the art, including those described herein. Specific illustrative and exemplary embodiments for measuring binding affinity are described in the following.

An “affinity matured” antibody refers to an antibody with one or more alterations in one or more hypervariable regions (HVRs), compared to a parent antibody which does not possess such alterations, such alterations resulting in an improvement in the affinity of the antibody for antigen.

The terms “anti-Tau antibody” and “an antibody that binds to Tau” refer to an antibody that is capable of binding Tau with sufficient affinity such that the antibody is useful as a diagnostic and/or therapeutic agent in targeting Tau. In some embodiments, the extent of binding of an anti-Tau antibody to an unrelated, non-Tau protein is less than about 10% of the binding of the antibody to Tau as measured, e.g., by a radioimmunoassay (RIA). In certain embodiments, an antibody that binds to Tau has a dissociation constant (K_(D)) of ≤1 μM, ≤100 nM, ≤10 nM, ≤1 nM, ≤0.1 nM, ≤0.01 nM, or ≤0.001 nM (e.g. 10⁻⁸M or less, e.g. from 10⁻⁸ M to 10⁻¹³M, e.g., from 10⁻⁹M to 10⁻¹³M). In certain embodiments, an anti-Tau antibody binds to an epitope of Tau that is conserved among Tau from different species. The term “anti-Tau antibody” and “antibody that binds to Tau,” as used herein, refers to an antibody that binds monomeric Tau, oligomeric Tau, and/or phosphorylated Tau, unless specifically indicated otherwise. In some such embodiments, the anti-Tau antibody binds to monomeric Tau, oligomeric Tau, non-phosphorylated Tau, and phosphorylated Tau with comparable affinities, such as with affinities that differ by no more than 50-fold from one another. In some embodiments, an antibody that binds monomeric Tau, oligomeric Tau, non-phosphorylated Tau, and phosphorylated Tau is referred to as a “pan-Tau antibody.”

The term “antibody” herein is used in the broadest sense and encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments so long as they exhibit the desired antigen-binding activity.

An “antibody fragment” refers to a molecule other than an intact antibody that comprises a portion of an intact antibody that binds the antigen to which the intact antibody binds. Examples of antibody fragments include but are not limited to Fv, Fab, Fab′, Fab′-SH, F(ab′)₂; diabodies; linear antibodies; single-chain antibody molecules (e.g. scFv); and multispecific antibodies formed from antibody fragments.

An “antibody that binds to the same epitope” as a reference antibody refers to an antibody that blocks binding of the reference antibody to its antigen in a competition assay by 50% or more, and conversely, the reference antibody blocks binding of the antibody to its antigen in a competition assay by 50% or more. An exemplary competition assay is provided herein.

The term “chimeric” antibody refers to an antibody in which a portion of the heavy and/or light chain is derived from a particular source or species, while the remainder of the heavy and/or light chain is derived from a different source or species.

The “class” of an antibody refers to the type of constant domain or constant region possessed by its heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into subclasses (isotypes), e.g., IgG₁, IgG₂, IgG₃, IgG₄, IgA₁, and IgA₂. The heavy chain constant domains that correspond to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

The term “cytotoxic agent” as used herein refers to a substance that inhibits or prevents a cellular function and/or causes cell death or destruction. Cytotoxic agents include, but are not limited to, radioactive isotopes (e.g., At²¹¹, I¹³¹, I¹²⁵, Y⁹⁰, Re¹⁸⁶, Re¹⁸⁸, Sm¹⁵³, Bi²¹², P³², Pb²¹² and radioactive isotopes of Lu); chemotherapeutic agents or drugs (e.g., methotrexate, adriamicin, vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin or other intercalating agents); growth inhibitory agents; enzymes and fragments thereof such as nucleolytic enzymes; antibiotics; toxins such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including fragments and/or variants thereof; and the various antitumor or anticancer agents disclosed below.

“Effector functions” refer to those biological activities attributable to the Fc region of an antibody, which vary with the antibody isotype. Examples of antibody effector functions include: C1q binding and complement dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; down regulation of cell surface receptors (e.g. B cell receptor); and B cell activation.

An “effective amount” of an agent, e.g., a pharmaceutical formulation, refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result.

The term “Fc region” herein is used to define a C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region. The term includes native sequence Fc regions and variant Fc regions. In some embodiments, a human IgG heavy chain Fc region extends from Cys226, or from Pro230, to the carboxyl-terminus of the heavy chain. However, the C-terminal lysine (Lys447) of the Fc region may or may not be present. Unless otherwise specified herein, numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also called the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991.

“Framework” or “FR” refers to variable domain residues other than hypervariable region (HVR) residues. The FR of a variable domain generally consists of four FR domains: FR1, FR2, FR3, and FR4. Accordingly, the HVR and FR sequences generally appear in the following sequence in VH (or VL): FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

The terms “full length antibody,” “intact antibody,” and “whole antibody” are used herein interchangeably to refer to an antibody having a structure substantially similar to a native antibody structure or having heavy chains that contain an Fc region as defined herein.

The terms “host cell,” “host cell line,” and “host cell culture” are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include “transformants” and “transformed cells,” which include the primary transformed cell and progeny derived therefrom without regard to the number of passages. Progeny may not be completely identical in nucleic acid content to a parent cell, but may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected for in the originally transformed cell are included herein.

A “human antibody” is one which possesses an amino acid sequence which corresponds to that of an antibody produced by a human or a human cell or derived from a non-human source that utilizes human antibody repertoires or other human antibody-encoding sequences. This definition of a human antibody specifically excludes a humanized antibody comprising non-human antigen-binding residues.

The term “variable region” or “variable domain” refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to antigen. The variable domains of the heavy chain and light chain (VH and VL, respectively) of a native antibody generally have similar structures, with each domain comprising four conserved framework regions (FRs) and three hypervariable regions (HVRs). (See, e.g., Kindt et al. Kuby Immunology, 6^(th) ed., W.H. Freeman and Co., page 91 (2007).) A single VH or VL domain may be sufficient to confer antigen-binding specificity. Furthermore, antibodies that bind a particular antigen may be isolated using a VH or VL domain from an antibody that binds the antigen to screen a library of complementary VL or VH domains, respectively. See, e.g., Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).

A “human consensus framework” is a framework which represents the most commonly occurring amino acid residues in a selection of human immunoglobulin VL or VH framework sequences. Generally, the selection of human immunoglobulin VL or VH sequences is from a subgroup of variable domain sequences. Generally, the subgroup of sequences is a subgroup as in Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication 91-3242, Bethesda Md. (1991), vols. 1-3. In some embodiments, for the VL, the subgroup is subgroup kappa I as in Kabat et al., supra. In some embodiments, for the VH, the subgroup is subgroup III as in Kabat et al., supra.

A “humanized” antibody refers to a chimeric antibody comprising amino acid residues from non-human HVRs and amino acid residues from human FRs. In certain embodiments, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the HVRs (e.g., CDRs) correspond to those of a non-human antibody, and all or substantially all of the FRs correspond to those of a human antibody. A humanized antibody optionally may comprise at least a portion of an antibody constant region derived from a human antibody. A “humanized form” of an antibody, e.g., a non-human antibody, refers to an antibody that has undergone humanization.

The term “hypervariable region” or “HVR” as used herein refers to each of the regions of an antibody variable domain which are hypervariable in sequence (“complementarity determining regions” or “CDRs”) and/or form structurally defined loops (“hypervariable loops”) and/or contain the antigen-contacting residues (“antigen contacts”). Generally, antibodies comprise six HVRs: three in the VH (H1, H2, H3), and three in the VL (L1, L2, L3). Exemplary HVRs herein include:

(a) hypervariable loops occurring at amino acid residues 26-32 (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101 (H3) (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987));

(b) CDRs occurring at amino acid residues 24-34 (L1), 50-56 (L2), 89-97 (L3), 31-35b (H1), 50-65 (H2), and 95-102 (H3) (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991));

(c) antigen contacts occurring at amino acid residues 27c-36 (L1), 46-55 (L2), 89-96 (L3), 30-35b (H1), 47-58 (H2), and 93-101 (H3) (MacCallum et al. J. Mol. Biol. 262: 732-745 (1996)); and

(d) combinations of (a), (b), and/or (c), including HVR amino acid residues 46-56 (L2), 47-56 (L2), 48-56 (L2), 49-56 (L2), 26-35 (H1), 26-35b (H1), 49-65 (H2), 93-102 (H3), and 94-102 (H3).

Unless otherwise indicated, HVR residues and other residues in the variable domain (e.g., FR residues) are numbered herein according to Kabat et al., supra.

An “immunoconjugate” is an antibody conjugated to one or more heterologous molecule(s), including but not limited to a cytotoxic agent.

An “individual” or “subject” is a mammal. Mammals include, but are not limited to, domesticated animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In certain embodiments, the individual or subject is a human.

An “isolated” antibody is one which has been separated from a component of its natural environment. In some embodiments, an antibody is purified to greater than 95% or 99% purity as determined by, for example, electrophoretic (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatographic (e.g., ion exchange or reverse phase HPLC). For review of methods for assessment of antibody purity, see, e.g., Flatman et al., J. Chromatogr. B 848:79-87 (2007).

An “isolated” nucleic acid refers to a nucleic acid molecule that has been separated from a component of its natural environment. An isolated nucleic acid includes a nucleic acid molecule contained in cells that ordinarily contain the nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location.

“Isolated nucleic acid encoding an anti-Tau antibody” refers to one or more nucleic acid molecules encoding antibody heavy and light chains (or fragments thereof), including such nucleic acid molecule(s) in a single vector or separate vectors, and such nucleic acid molecule(s) present at one or more locations in a host cell.

The term “monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical and/or bind the same epitope, except for possible variant antibodies, e.g., containing naturally occurring mutations or arising during production of a monoclonal antibody preparation, such variants generally being present in minor amounts. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen. Thus, the modifier “monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the present invention may be made by a variety of techniques, including but not limited to the hybridoma method, recombinant DNA methods, phage-display methods, and methods utilizing transgenic animals containing all or part of the human immunoglobulin loci, such methods and other exemplary methods for making monoclonal antibodies being described herein.

A “naked antibody” refers to an antibody that is not conjugated to a heterologous moiety (e.g., a cytotoxic moiety) or radiolabel. The naked antibody may be present in a pharmaceutical formulation.

“Native antibodies” refer to naturally occurring immunoglobulin molecules with varying structures. For example, native IgG antibodies are heterotetrameric glycoproteins of about 150,000 daltons, composed of two identical light chains and two identical heavy chains that are disulfide-bonded. From N- to C-terminus, each heavy chain has a variable region (VH), also called a variable heavy domain or a heavy chain variable domain, followed by three constant domains (CH1, CH2, and CH3). Similarly, from N- to C-terminus, each light chain has a variable region (VL), also called a variable light domain or a light chain variable domain, followed by a constant light (CL) domain. The light chain of an antibody may be assigned to one of two types, called kappa (κ) and lambda (λ), based on the amino acid sequence of its constant domain.

The term “package insert” is used to refer to instructions customarily included in commercial packages of therapeutic products, that contain information about the indications, usage, dosage, administration, combination therapy, contraindications and/or warnings concerning the use of such therapeutic products.

“Percent (%) amino acid sequence identity” with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. For purposes herein, however, % amino acid sequence identity values are generated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program was authored by Genentech, Inc., and the source code has been filed with user documentation in the U.S. Copyright Office, Washington D.C., 20559, where it is registered under U.S. Copyright Registration No. TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc., South San Francisco, Calif., or may be compiled from the source code. The ALIGN-2 program should be compiled for use on a UNIX operating system, including digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not vary.

In situations where ALIGN-2 is employed for amino acid sequence comparisons, the % amino acid sequence identity of a given amino acid sequence A to, with, or against a given amino acid sequence B (which can alternatively be phrased as a given amino acid sequence A that has or comprises a certain % amino acid sequence identity to, with, or against a given amino acid sequence B) is calculated as follows:

100 times the fraction X/Y

where X is the number of amino acid residues scored as identical matches by the sequence alignment program ALIGN-2 in that program's alignment of A and B, and where Y is the total number of amino acid residues in B. It will be appreciated that where the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity of A to B will not equal the % amino acid sequence identity of B to A. Unless specifically stated otherwise, all % amino acid sequence identity values used herein are obtained as described in the immediately preceding paragraph using the ALIGN-2 computer program.

The term “pharmaceutical formulation” refers to a preparation which is in such form as to permit the biological activity of an active ingredient contained therein to be effective, and which contains no additional components which are unacceptably toxic to a subject to which the formulation would be administered.

A “pharmaceutically acceptable carrier” refers to an ingredient in a pharmaceutical formulation, other than an active ingredient, which is nontoxic to a subject. A pharmaceutically acceptable carrier includes, but is not limited to, a buffer, excipient, stabilizer, or preservative.

The term “Tau,” as used herein, refers to any native Tau protein from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats), unless otherwise indicated. The term encompasses “full-length,” unprocessed Tau as well as any form of Tau that results from processing in the cell. The term also encompasses naturally occurring variants of Tau, e.g., splice variants or allelic variants.

The term “pTau,” as used herein, refers to Tau in which a serine, a threonine or a tyrosine residue is phosphorylated by a protein kinase by the addition of a covalently bound phosphate group. In some embodiments, pTau is phosphorylated on a serine or on a threonine residue. In some embodiments, pTau is phosphorylated on Serine at position 409 and/or Serine at position 404. In some embodiments, pTau is phosphorylated on Serine at position 409.

The terms “soluble Tau” or “soluble Tau protein,” as used herein, refer to proteins consisting of both completely solubilized Tau protein/peptide monomers or of Tau-like peptides/proteins, or of modified or truncated Tau peptides/proteins or of other derivates of Tau peptides/proteins monomers, and of Tau protein oligomers. “Soluble Tau” excludes particularly neurofibrillary tangles (NFT).

The term “insoluble Tau,” as used herein, refers to multiple aggregated monomers of Tau peptides or proteins, or of Tau-like peptides/proteins, or of modified or truncated Tau peptides/proteins or of other derivates of Tau peptides/proteins forming oligomeric or polymeric structures which are insoluble both in vitro in aqueous medium and in vivo in the mammalian or human body more particularly in the brain, but particularly to multiple aggregated monomers of Tau or of modified or truncated Tau peptides/proteins or of derivatives thereof, which are insoluble in the mammalian or human body more particularly in the brain, respectively. “Insoluble Tau” particularly includes neurofibrillary tangles (NFT).

The terms “monomeric Tau” or “Tau monomer,” as used herein, refer to completely solubilized Tau proteins without aggregated complexes in aqueous medium.

The terms “aggregated Tau”, “oligomeric Tau” and “Tau oligomer,” as used herein, refer to multiple aggregated monomers of Tau peptides or proteins, or of Tau-like peptides/proteins, or of modified or truncated Tau peptides/proteins or of other derivates of Tau peptides/proteins forming oligomeric or polymeric structures which are insoluble or soluble both in vitro in aqueous medium and in vivo in the mammalian or human body more particularly in the brain, but particularly to multiple aggregated monomers of Tau or of modified or truncated Tau peptides/proteins or of derivatives thereof, which are insoluble or soluble in the mammalian or human body more particularly in the brain, respectively.

The terms “pTau PHF”, “PHF”, and “paired helical filaments,” are used herein synonymously, refer to pairs of filaments wound into helices with a periodicity of 160 nm visible on electron microscopy. Width varies between 10 and 22 nm. PHF are the predominant structures in neurofibrillary tangles of Alzheimer's Disease (AD) and neuropil threads. PHF may also be seen in some but not all dystrophic neurites associated with neuritic plaques. The major component of PHF is a hyperphosphorylated form of microtubule-associated protein tau. PHF may be partially composed of disulfide-linked antiparallel hyper-phosphorylated Tau proteins. PHF Tau may be truncated of its C-terminal 20 amino acid residues. The mechanisms underlying PHF formation are uncertain but hyper-phosphorylation of Tau may disengage it from microtubules, increasing the soluble pool of Tau from which PHF can be formed inside neurons.

As used herein, “treatment” (and grammatical variations thereof such as “treat” or “treating”) refers to clinical intervention in an attempt to alter the natural course of the individual being treated, and can be performed either for prophylaxis or during the course of clinical pathology. Desirable effects of treatment include, but are not limited to, preventing occurrence or recurrence of disease, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, preventing metastasis, decreasing the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis. In some embodiments, antibodies of the invention are used to delay development of a disease or to slow the progression of a disease.

The term “early Alzheimer's Disease” or “early AD” as used herein (e.g., a “patient diagnosed with early AD” or a “patient suffering from early AD”) includes patients with mild cognitive impairement, such as a memory deficit, due to AD and patients having AD biomarkers, for example amyloid positive patients.

The term “mild Alzheimer's Disease” or “mild AD” as used herein (e.g., a “patient diagnosed with mild AD”) refers to a stage of AD characterized by an MMSE score of 20 to 26.

The term “mild to moderate Alzheimer's Disease” or “mild to moderate AD” as used herein encompasses both mild and moderate AD, and is characterized by an MMSE score of 18 to 26.

The term “moderate Alzheimer's Disease” or “moderate AD” as used herein (e.g., a “patient diagnosed with moderate AD”) refers to a stage of AD characterized by an MMSE score of 18 to 19.

The term “MMSE” refers to the Mini Mental State Examination, which provides a score between 1 and 30. See Folstein, et al., 1975, J. Psychiatr. Res. 12:189-98. Scores of 26 and lower are generally considered to be indicative of a deficit. The lower the numerical score on the MMSE, the greater the tested patient's deficit or impairment relative to another individual with a lower score. An increase in MMSE score may be indicative of improvement in the patient's condition, whereas a decrease in MMSE score may denote worsening in the patient's condition.

The term “vector,” as used herein, refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes the vector as a self-replicating nucleic acid structure as well as the vector incorporated into the genome of a host cell into which it has been introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as “expression vectors.”

II. Compositions and Methods

Antibodies that bind Tau are provided. In some embodiments, an antibody provided herein binds human monomeric Tau with a K_(D) of less than 1 nM, or less than 0.5 nM. In some embodiments, an antibody provided herein binds cynomolgus monkey Tau with a K_(D) of less than 1 nM, or less than 0.5 nM. In some embodiments, K_(D) is determined by surface plasmon resonance at 37° C. In some embodiments, an antibody of the invention binds Tau binds monomeric Tau, oligomeric Tau, non-phosphorylated Tau, and phosphorylated Tau. In some embodiments, an antibody of the invention binds to an epitope within amino acids 2 to 24 of mature human Tau. In some embodiments, an antibody of the invention binds to an epitope within Tau amino acids 2 to 24 and binds monomeric Tau, oligomeric Tau, non-phosphorylated Tau, and phosphorylated Tau. In some embodiments, an antibody binds an epitope of human Tau having, or consisting of, the sequence AEPRQEFEVMEDHAGTYGLGDRK (SEQ ID NO: 2). In some embodiments, an antibody binds an epitope of cynomolgus monkey Tau having, or consisting of, the sequence AEPRQEFDVMEDHAGTYGLGDRK (SEQ ID NO: 4). In some embodiments, an antibody binds an epitope of human Tau having, or consisting of, the sequence AEPRQEFEVMEDHAGTYGLGDRK (SEQ ID NO: 2) and an epitope of cynomolgus monkey Tau having, or consisting of, the sequence AEPRQEFDVMEDHAGTYGLGDRK (SEQ ID NO: 4).

In some embodiments, an antibody provided herein binds to an epitope within amino acids 19 to 33, 19 to 42, 37 to 51, 100 to 114, 118 to 132, or 172 to 177 of mature human Tau. In some embodiments, an antibody of the invention binds to an epitope within amino acids 19 to 33, 19 to 42, 37 to 51, 100 to 114, 118 to 132, or 172 to 177 of mature human Tau and binds monomeric Tau, oligomeric Tau, non-phosphorylated Tau, and phosphorylated Tau.

Antibodies of the invention are useful, e.g., for the diagnosis or treatment of neurodegenerative diseases.

A. Exemplary Anti-Tau Antibodies

In some embodiments, the antibody comprises HVR-H1 comprising an amino acid sequence of SEQ ID NO: 605; HVR-H2 comprising an amino acid sequence of SEQ ID NO: 606; and HVR-H3 comprising an amino acid sequence of SEQ ID NO: 607.

In some embodiments, the antibody comprises HVR-L1 comprising an amino acid sequence of SEQ ID NO: 608; HVR-L2 comprising an amino acid sequence of SEQ ID NO: 609; and HVR-L3 comprising an amino acid sequence of SEQ ID NO: 610.

In some embodiments, the antibody comprises HVR-H1 comprising an amino acid sequence of SEQ ID NO: 605; HVR-H2 comprising an amino acid sequence of SEQ ID NO: 606; HVR-H3 comprising an amino acid sequence of SEQ ID NO: 607; HVR-L1 comprising an amino acid sequence of SEQ ID NO: 608; HVR-L2 comprising an amino acid sequence of SEQ ID NO: 609; and HVR-L3 comprising an amino acid sequence of SEQ ID NO: 610.

In any of the above embodiments, an anti-Tau antibody is humanized. In some embodiments, an anti-Tau antibody comprises HVRs as in any of the above embodiments, and further comprises an acceptor human framework, e.g. a human immunoglobulin framework or a human consensus framework.

In another aspect, an anti-Tau antibody comprises a heavy chain variable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 340, 603, 614, or 619. In certain embodiments, a VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but an anti-Tau antibody comprising that sequence retains the ability to bind to Tau. In certain embodiments, a total of 1 to 10 amino acids have been substituted, inserted and/or deleted in SEQ ID NO: 340, 603, 614, or 619. In certain embodiments, substitutions, insertions, or deletions occur in regions outside the HVRs (i.e., in the FRs). Optionally, the anti-Tau antibody comprises the VH sequence in SEQ ID NO: 340, 603, 614, or 619, including post-translational modifications of that sequence. In a particular embodiment, the VH comprises one, two or three HVRs selected from: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 605, (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 606, and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 607.

In another aspect, an anti-Tau antibody comprises a light chain variable domain (VL) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 341, 604, 615, or 620. In certain embodiments, a VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but an anti-Tau antibody comprising that sequence retains the ability to bind to Tau. In certain embodiments, a total of 1 to 10 amino acids have been substituted, inserted and/or deleted in SEQ ID NO: 341, 604, 615, or 620. In certain embodiments, substitutions, insertions, or deletions occur in regions outside the HVRs (i.e., in the FRs). Optionally, the anti-Tau antibody comprises the VL sequence in SEQ ID NO: 341, 604, 615, or 620, including post-translational modifications of that sequence. In a particular embodiment, the VL comprises one, two or three HVRs selected from: (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 608, (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 609, and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 610.

In some embodiments, an anti-Tau antibody comprises:

-   -   a) a heavy chain variable region (VH) comprising a sequence that         is at least 95% identical to SEQ ID NO: 603;     -   b) a light chain variable region (VL) comprising a sequence that         is at least 95% identical to SEQ ID NO: 604;     -   c) a VH as in (a) and a VL as in (b);     -   d) a heavy chain variable region (VH) comprising a sequence that         is at least 95% identical to SEQ ID NO: 614;     -   e) a light chain variable region (VL) comprising a sequence that         is at least 95% identical to SEQ ID NO: 615;     -   f) a VH as in (d) and a VL as in (e);     -   g) a heavy chain variable region (VH) comprising a sequence that         is at least 95% identical to SEQ ID NO: 619;     -   h) a light chain variable region (VL) comprising a sequence that         is at least 95% identical to SEQ ID NO: 620;     -   i) a VH as in (g) and a VL as in (h).

In some embodiments, an anti-Tau antibody comprises:

-   -   a) a heavy chain variable region (VH) comprising SEQ ID NO: 603;     -   b) a light chain variable region (VL) comprising SEQ ID NO: 604;     -   c) a VH as in (a) and a VL as in (b);     -   d) a heavy chain variable region (VH) comprising the sequence of         SEQ ID NO: 614;     -   e) a light chain variable region (VL) comprising the sequence of         SEQ ID NO: 615;     -   f) a VH as in (d) and a VL as in (e);     -   g) a heavy chain variable region (VH) comprising the sequence of         SEQ ID NO: 619;     -   h) a light chain variable region (VL) comprising the sequence of         SEQ ID NO: 620;     -   i) a VH as in (g) and a VL as in (h).

In some embodiments, an anti-Tau antibody comprises a heavy chain variable region comprising a sequence selected from SEQ ID NOs: 603, 614, and 619; and a light chain variable region comprising a sequence selected from SEQ ID NOs: 604, 615, and 620. In some embodiments, the antibody comprises a heavy chain variable region comprising a sequence selected from SEQ ID NOs: 340, 603, 614, and 619; and a light chain variable region comprising a sequence selected from SEQ ID NOs: 604, 615, and 620. In some embodiments, the antibody comprises a heavy chain variable region comprising a sequence selected from SEQ ID NOs: 603, 614, and 619; and a light chain variable region comprising a sequence selected from SEQ ID NOs: 341, 604, 615, and 620.

In some embodiments, an anti-Tau antibody comprises (a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 603 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 604; (b) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 614 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 615; or (c) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 619 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 620.

In some embodiments, an anti-Tau antibody comprises (a) a heavy chain comprising the amino acid sequence of SEQ ID NO: 611 or SEQ ID NO: 612 and a light chain comprising the amino acid sequence of SEQ ID NO: 613; (b) a heavy chain comprising the amino acid sequence of SEQ ID NO: 616 or SEQ ID NO: 617 and a light chain comprising the amino acid sequence of SEQ ID NO: 618; or (c) a heavy chain comprising the amino acid sequence of SEQ ID NO: 621 or SEQ ID NO: 622 and a light chain comprising the amino acid sequence of SEQ ID NO: 623.

In some embodiments, an isolated antibody that binds to human Tau is provided, wherein the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 611 or SEQ ID NO: 612 and a light chain comprising the amino acid sequence of SEQ ID NO: 613. In some embodiments, an isolated antibody that binds to human Tau is provided, wherein the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 611 and a light chain comprising the amino acid sequence of SEQ ID NO: 613. In some embodiments, an isolated antibody that binds to human Tau is provided, wherein the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 612 and a light chain comprising the amino acid sequence of SEQ ID NO: 613. In some embodiments, an isolated antibody that binds to human Tau is provided, wherein the antibody comprises a heavy chain consisting of the amino acid sequence of SEQ ID NO: 611 or SEQ ID NO: 612 and a light chain consisting of the amino acid sequence of SEQ ID NO: 613. In some embodiments, an isolated antibody that binds to human Tau is provided, wherein the antibody comprises a heavy chain consisting of the amino acid sequence of SEQ ID NO: 611 and a light chain consisting of the amino acid sequence of SEQ ID NO: 613. In some embodiments, an isolated antibody that binds to human Tau is provided, wherein the antibody comprises a heavy chain consisting of the amino acid sequence of SEQ ID NO: 612 and a light chain consisting of the amino acid sequence of SEQ ID NO: 613.

In some embodiments, an isolated antibody that binds to human Tau is provided, wherein the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 616 or 617 and a light chain comprising the amino acid sequence of SEQ ID NO: 618. In some embodiments, an isolated antibody that binds to human Tau is provided, wherein the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 616 and a light chain comprising the amino acid sequence of SEQ ID NO: 618. In some embodiments, an isolated antibody that binds to human Tau is provided, wherein the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 617 and a light chain comprising the amino acid sequence of SEQ ID NO: 618. In some embodiments, an isolated antibody that binds to human Tau is provided, wherein the antibody comprises a heavy chain consisting of the amino acid sequence of SEQ ID NO: 616 or SEQ ID NO: 617 and a light chain consisting of the amino acid sequence of SEQ ID NO: 618. In some embodiments, an isolated antibody that binds to human Tau is provided, wherein the antibody comprises a heavy chain consisting of the amino acid sequence of SEQ ID NO: 616 and a light chain consisting of the amino acid sequence of SEQ ID NO: 618. In some embodiments, an isolated antibody that binds to human Tau is provided, wherein the antibody comprises a heavy chain consisting of the amino acid sequence of SEQ ID NO: 617 and a light chain consisting of the amino acid sequence of SEQ ID NO: 618.

In some embodiments, an isolated antibody that binds to human Tau is provided, wherein the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 621 or SEQ ID NO: 622 and a light chain comprising the amino acid sequence of SEQ ID NO: 623. In some embodiments, an isolated antibody that binds to human Tau is provided, wherein the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 621 and a light chain comprising the amino acid sequence of SEQ ID NO: 623. In some embodiments, an isolated antibody that binds to human Tau is provided, wherein the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 622 and a light chain comprising the amino acid sequence of SEQ ID NO: 623. In some embodiments, an isolated antibody that binds to human Tau is provided, wherein the antibody comprises a heavy chain consisting of the amino acid sequence of SEQ ID NO: 621 or SEQ ID NO: 622 and a light chain consisting of the amino acid sequence of SEQ ID NO: 623. In some embodiments, an isolated antibody that binds to human Tau is provided, wherein the antibody comprises a heavy chain consisting of the amino acid sequence of SEQ ID NO: 621 and a light chain consisting of the amino acid sequence of SEQ ID NO: 623. In some embodiments, an isolated antibody that binds to human Tau is provided, wherein the antibody comprises a heavy chain consisting of the amino acid sequence of SEQ ID NO: 622 and a light chain consisting of the amino acid sequence of SEQ ID NO: 623.

In a further aspect, the invention provides an antibody that binds to the same epitope as an anti-Tau antibody provided herein. For example, in certain embodiments, an antibody is provided that binds to the same epitope as an antibody selected from 37D3-H9, hu37D3-H9.v28.A4, hu37D3-H9.v76, hu37D3-H9.v83, and hu37D3-H9.v93. In certain embodiments, an antibody is provided that binds to an epitope within a fragment of Tau consisting of amino acids 2-24 of SEQ ID NO: 2. In certain embodiments, an antibody is provided that binds to an epitope within a fragment of Tau consisting of amino acids 7-24 of SEQ ID NO: 2. In certain embodiments, an antibody is provided that binds to an epitope within a fragment of Tau consisting of amino acids 7-20 of SEQ ID NO: 2. In certain embodiments, an antibody is provided that binds to an epitope within a fragment of Tau consisting of amino acids 10-24 of SEQ ID NO: 2. In certain embodiments, an antibody is provided that binds to an epitope within a fragment of Tau consisting of amino acids 7-21 of SEQ ID NO: 2. In certain embodiments, an antibody is provided that binds to an epitope within a fragment of Tau consisting of amino acids 8-22 of SEQ ID NO: 2. In certain embodiments, an antibody is provided that binds to an epitope within a fragment of Tau consisting of amino acids 11-25 of SEQ ID NO: 2. In certain embodiments, an antibody is provided that binds to one or more, or all, of the following fragments of Tau: 2-24, 7-24, 7-20, 10-24, 7-21, 8-22, and 11-25. In some embodiments, an antibody is provided that binds to a peptide having the sequence of SEQ ID NO: 593, but does not bind to a peptide having the sequence of SEQ ID NO: 596 or SEQ ID NO: 597.

In a further aspect of the invention, an anti-Tau antibody according to any of the above embodiments is a monoclonal antibody, including a chimeric, humanized or human antibody. In one embodiment, an anti-Tau antibody is an antibody fragment, e.g., a Fv, Fab, Fab′, scFv, diabody, or F(ab′)2 fragment. In another embodiment, the antibody is a full length antibody, e.g., an intact IgG1 or IgG4 antibody or other antibody class or isotype as defined herein.

In a further aspect, an anti-Tau antibody according to any of the above embodiments may incorporate any of the features, singly or in combination, as described in Sections 1-7 below:

1. Antibody Affinity

In certain embodiments, an antibody provided herein has a dissociation constant (K_(D)) of ≤1 μM, ≤100 nM, ≤10 nM, ≤1 nM, ≤0.1 nM, ≤0.01 nM, or ≤0.001 nM (e.g. 10⁻⁸M or less, e.g. from 10⁻⁸M to 10⁻¹³M, e.g., from 10⁻⁹M to 10⁻¹³M).

In some embodiments, K_(D) is measured by a radiolabeled antigen binding assay (RIA). In some embodiments, an RIA is performed with the Fab version of an antibody of interest and its antigen. For example, solution binding affinity of Fabs for antigen is measured by equilibrating Fab with a minimal concentration of (¹²⁵I)-labeled antigen in the presence of a titration series of unlabeled antigen, then capturing bound antigen with an anti-Fab antibody-coated plate (see, e.g., Chen et al., J. Mol. Biol. 293:865-881(1999)). To establish conditions for the assay, MICROTITER® multi-well plates (Thermo Scientific) are coated overnight with 5 μg/ml of a capturing anti-Fab antibody (Cappel Labs) in 50 mM sodium carbonate (pH 9.6), and subsequently blocked with 2% (w/v) bovine serum albumin in PBS for two to five hours at room temperature (approximately 23° C.). In a non-adsorbent plate (Nunc #269620), 100 pM or 26 pM [¹²⁵I]-antigen are mixed with serial dilutions of a Fab of interest (e.g., consistent with assessment of the anti-VEGF antibody, Fab-12, in Presta et al., Cancer Res. 57:4593-4599 (1997)). The Fab of interest is then incubated overnight; however, the incubation may continue for a longer period (e.g., about 65 hours) to ensure that equilibrium is reached. Thereafter, the mixtures are transferred to the capture plate for incubation at room temperature (e.g., for one hour). The solution is then removed and the plate washed eight times with 0.1% polysorbate 20 (TWEEN-20) in PBS. When the plates have dried, 150 μl/well of scintillant (MICROSCINT-20™; Packard) is added, and the plates are counted on a TOPCOUNT™ gamma counter (Packard) for ten minutes. Concentrations of each Fab that give less than or equal to 20% of maximal binding are chosen for use in competitive binding assays.

According to another embodiment, K_(D) is measured using a BIACORE® surface plasmon resonance assay. For example, an assay using a BIACORE®-2000 or a BIACORE®-3000 (BIAcore, Inc., Piscataway, N.J.) is performed at 25° C. or 37° C. with immobilized antigen CM5 chips at ˜10 resonance units (RU). In some embodiments, carboxymethylated dextran biosensor chips (CM5, BIACORE, Inc.) are activated with N-ethyl-N′-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NETS) according to the supplier's instructions. Antigen is diluted with 10 mM sodium acetate, pH 4.8, to 5 μg/ml (˜0.2 μM) before injection at a flow rate of 5 μl/minute to achieve approximately 10 resonance units (RU) of coupled protein. Following the injection of antigen, 1 M ethanolamine is injected to block unreacted groups. For kinetics measurements, two-fold serial dilutions of Fab (0.78 nM to 500 nM) are injected in PBS with 0.05% polysorbate 20 (TWEEN-20™) surfactant (PBST) at 25° C. at a flow rate of approximately 25 μl/min. Association rates (k_(on)) and dissociation rates (k_(off)) are calculated using a simple one-to-one Langmuir binding model (BIACORE® Evaluation Software version 3.2) by simultaneously fitting the association and dissociation sensorgrams. The equilibrium dissociation constant (K_(D)) is calculated as the ratio k_(off)/k_(on). See, e.g., Chen et al., J. Mol. Biol. 293:865-881 (1999). If the on-rate exceeds 10⁶ M⁻¹ s⁻¹ by the surface plasmon resonance assay above, then the on-rate can be determined by using a fluorescent quenching technique that measures the increase or decrease in fluorescence emission intensity (excitation=295 nm; emission=340 nm, 16 nm band-pass) at 25° C. of a 20 nM anti-antigen antibody (Fab form) in PBS, pH 7.2, in the presence of increasing concentrations of antigen as measured in a spectrometer, such as a stop-flow equipped spectrophometer (Aviv Instruments) or a 8000-series SLM-AMINCO™ spectrophotometer (ThermoSpectronic) with a stirred cuvette.

2. Antibody Fragments

In certain embodiments, an antibody provided herein is an antibody fragment. Antibody fragments include, but are not limited to, Fab, Fab′, Fab′-SH, F(ab′)₂, Fv, and scFv fragments, and other fragments described below. For a review of certain antibody fragments, see Hudson et al. Nat. Med. 9:129-134 (2003). For a review of scFv fragments, see, e.g., Pluckthün, in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., (Springer-Verlag, New York), pp. 269-315 (1994); see also WO 93/16185; and U.S. Pat. Nos. 5,571,894 and 5,587,458. For discussion of Fab and F(ab′)2 fragments comprising salvage receptor binding epitope residues and having increased in vivo half-life, see U.S. Pat. No. 5,869,046.

Diabodies are antibody fragments with two antigen-binding sites that may be bivalent or bispecific. See, for example, EP 404,097; WO 1993/01161; Hudson et al., Nat. Med. 9:129-134 (2003); and Hollinger et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993). Triabodies and tetrabodies are also described in Hudson et al., Nat. Med. 9:129-134 (2003).

Single-domain antibodies are antibody fragments comprising all or a portion of the heavy chain variable domain or all or a portion of the light chain variable domain of an antibody. In certain embodiments, a single-domain antibody is a human single-domain antibody (Domantis, Inc., Waltham, Mass.; see, e.g., U.S. Pat. No. 6,248,516 B 1).

Antibody fragments can be made by various techniques, including but not limited to proteolytic digestion of an intact antibody as well as production by recombinant host cells (e.g. E. coli or phage), as described herein.

3. Chimeric and Humanized Antibodies

In certain embodiments, an antibody provided herein is a chimeric antibody. Certain chimeric antibodies are described, e.g., in U.S. Pat. No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)). In one example, a chimeric antibody comprises a non-human variable region (e.g., a variable region derived from a mouse, rat, hamster, rabbit, or non-human primate, such as a monkey) and a human constant region. In a further example, a chimeric antibody is a “class switched” antibody in which the class or subclass has been changed from that of the parent antibody. Chimeric antibodies include antigen-binding fragments thereof.

In certain embodiments, a chimeric antibody is a humanized antibody. Typically, a non-human antibody is humanized to reduce immunogenicity to humans, while retaining the specificity and affinity of the parental non-human antibody. Generally, a humanized antibody comprises one or more variable domains in which HVRs, e.g., CDRs, (or portions thereof) are derived from a non-human antibody, and FRs (or portions thereof) are derived from human antibody sequences. A humanized antibody optionally will also comprise at least a portion of a human constant region. In some embodiments, some FR residues in a humanized antibody are substituted with corresponding residues from a non-human antibody (e.g., the antibody from which the HVR residues are derived), e.g., to restore or improve antibody specificity or affinity.

Humanized antibodies and methods of making them are reviewed, e.g., in Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008), and are further described, e.g., in Riechmann et al., Nature 332:323-329 (1988); Queen et al., Proc. Nat'l Acad. Sci. USA 86:10029-10033 (1989); U.S. Pat. Nos. 5,821,337, 7,527,791, 6,982,321, and 7,087,409; Kashmiri et al., Methods 36:25-34 (2005) (describing specificity determining region (SDR) grafting); Padlan, Mol. Immunol. 28:489-498 (1991) (describing “resurfacing”); Dall'Acqua et al., Methods 36:43-60 (2005) (describing “FR shuffling”); and Osbourn et al., Methods 36:61-68 (2005) and Klimka et al., Br. J. Cancer, 83:252-260 (2000) (describing the “guided selection” approach to FR shuffling).

Human framework regions that may be used for humanization include but are not limited to: framework regions selected using the “best-fit” method (see, e.g., Sims et al. J. Immunol. 151:2296 (1993)); framework regions derived from the consensus sequence of human antibodies of a particular subgroup of light or heavy chain variable regions (see, e.g., Carter et al. Proc. Natl. Acad. Sci. USA, 89:4285 (1992); and Presta et al. J. Immunol., 151:2623 (1993)); human mature (somatically mutated) framework regions or human germline framework regions (see, e.g., Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008)); and framework regions derived from screening FR libraries (see, e.g., Baca et al., J. Biol. Chem. 272:10678-10684 (1997) and Rosok et al., J. Biol. Chem. 271:22611-22618 (1996)).

4. Human Antibodies

In certain embodiments, an antibody provided herein is a human antibody. Human antibodies can be produced using various techniques known in the art. Human antibodies are described generally in van Dijk and van de Winkel, Curr. Opin. Pharmacol. 5: 368-74 (2001) and Lonberg, Curr. Opin. Immunol. 20:450-459 (2008).

Human antibodies may be prepared by administering an immunogen to a transgenic animal that has been modified to produce intact human antibodies or intact antibodies with human variable regions in response to antigenic challenge. Such animals typically contain all or a portion of the human immunoglobulin loci, which replace the endogenous immunoglobulin loci, or which are present extrachromosomally or integrated randomly into the animal's chromosomes. In such transgenic mice, the endogenous immunoglobulin loci have generally been inactivated. For review of methods for obtaining human antibodies from transgenic animals, see Lonberg, Nat. Biotech. 23:1117-1125 (2005). See also, e.g., U.S. Pat. Nos. 6,075,181 and 6,150,584 describing XENOMOUSE™ technology; U.S. Pat. No. 5,770,429 describing HuMAB® technology; U.S. Pat. No. 7,041,870 describing K-M MOUSE® technology, and U.S. Patent Application Publication No. US 2007/0061900, describing VELOCIMOUSE® technology). Human variable regions from intact antibodies generated by such animals may be further modified, e.g., by combining with a different human constant region.

Human antibodies can also be made by hybridoma-based methods. Human myeloma and mouse-human heteromyeloma cell lines for the production of human monoclonal antibodies have been described. (See, e.g., Kozbor J. Immunol., 133: 3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987); and Boerner et al., J. Immunol., 147: 86 (1991).) Human antibodies generated via human B-cell hybridoma technology are also described in Li et al., Proc. Natl. Acad. Sci. USA, 103:3557-3562 (2006). Additional methods include those described, for example, in U.S. Pat. No. 7,189,826 (describing production of monoclonal human IgM antibodies from hybridoma cell lines) and Ni, Xiandai Mianyixue, 26(4):265-268 (2006) (describing human-human hybridomas). Human hybridoma technology (Trioma technology) is also described in Vollmers and Brandlein, Histology and Histopathology, 20(3):927-937 (2005) and Vollmers and Brandlein, Methods and Findings in Experimental and Clinical Pharmacology, 27(3):185-91 (2005).

Human antibodies may also be generated by isolating Fv clone variable domain sequences selected from human-derived phage display libraries. Such variable domain sequences may then be combined with a desired human constant domain. Techniques for selecting human antibodies from antibody libraries are described below.

5. Library-Derived Antibodies

Antibodies of the invention may be isolated by screening combinatorial libraries for antibodies with the desired activity or activities. For example, a variety of methods are known in the art for generating phage display libraries and screening such libraries for antibodies possessing the desired binding characteristics. Such methods are reviewed, e.g., in Hoogenboom et al. in Methods in Molecular Biology 178:1-37 (O'Brien et al., ed., Human Press, Totowa, N.J., 2001) and further described, e.g., in the McCafferty et al., Nature 348:552-554; Clackson et al., Nature 352: 624-628 (1991); Marks et al., J. Mol. Biol. 222: 581-597 (1992); Marks and Bradbury, in Methods in Molecular Biology 248:161-175 (Lo, ed., Human Press, Totowa, N.J., 2003); Sidhu et al., J. Mol. Biol. 338(2): 299-310 (2004); Lee et al., J. Mol. Biol. 340(5): 1073-1093 (2004); Fellouse, Proc. Natl. Acad. Sci. USA 101(34): 12467-12472 (2004); and Lee et al., J. Immunol. Methods 284(1-2): 119-132(2004).

In certain phage display methods, repertoires of VH and VL genes are separately cloned by polymerase chain reaction (PCR) and recombined randomly in phage libraries, which can then be screened for antigen-binding phage as described in Winter et al., Ann. Rev. Immunol., 12: 433-455 (1994). Phage typically display antibody fragments, either as single-chain Fv (scFv) fragments or as Fab fragments. Libraries from immunized sources provide high-affinity antibodies to the immunogen without the requirement of constructing hybridomas. Alternatively, the naive repertoire can be cloned (e.g., from human) to provide a single source of antibodies to a wide range of non-self and also self antigens without any immunization as described by Griffiths et al., EMBO J, 12: 725-734 (1993). Finally, naive libraries can also be made synthetically by cloning unrearranged V-gene segments from stem cells, and using PCR primers containing random sequence to encode the highly variable CDR3 regions and to accomplish rearrangement in vitro, as described by Hoogenboom and Winter, J. Mol. Biol., 227: 381-388 (1992). Patent publications describing human antibody phage libraries include, for example: U.S. Pat. No. 5,750,373, and US Patent Publication Nos. 2005/0079574, 2005/0119455, 2005/0266000, 2007/0117126, 2007/0160598, 2007/0237764, 2007/0292936, and 2009/0002360.

Antibodies or antibody fragments isolated from human antibody libraries are considered human antibodies or human antibody fragments herein.

6. Multispecific Antibodies

In certain embodiments, an antibody provided herein is a multispecific antibody, e.g. a bispecific antibody. Multispecific antibodies are monoclonal antibodies that have binding specificities for at least two different sites. In certain embodiments, one of the binding specificities is for Tau and the other is for any other antigen. In certain embodiments, one of the binding specificities is for Tau and the other is for amyloid beta. In certain embodiments, bispecific antibodies may bind to two different epitopes of Tau. Bispecific antibodies may also be used to localize cytotoxic agents to cells which express Tau. Bispecific antibodies can be prepared as full length antibodies or antibody fragments.

Techniques for making multispecific antibodies include, but are not limited to, recombinant co-expression of two immunoglobulin heavy chain-light chain pairs having different specificities (see Milstein and Cuello, Nature 305: 537 (1983)), WO 93/08829, and Traunecker et al., EMBO J. 10: 3655 (1991)), and “knob-in-hole” engineering (see, e.g., U.S. Pat. No. 5,731,168). Multi-specific antibodies may also be made by engineering electrostatic steering effects for making antibody Fc-heterodimeric molecules (WO 2009/089004A1); cross-linking two or more antibodies or fragments (see, e.g., U.S. Pat. No. 4,676,980, and Brennan et al., Science, 229: 81 (1985)); using leucine zippers to produce bi-specific antibodies (see, e.g., Kostelny et al., J. Immunol., 148(5):1547-1553 (1992)); using “diabody” technology for making bispecific antibody fragments (see, e.g., Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993)); and using single-chain Fv (sFv) dimers (see, e.g. Gruber et al., J. Immunol., 152:5368 (1994)); and preparing trispecific antibodies as described, e.g., in Tutt et al. J. Immunol. 147: 60 (1991).

Engineered antibodies with three or more functional antigen binding sites, including “Octopus antibodies,” are also included herein (see, e.g. US 2006/0025576A1).

The antibody or fragment herein also includes a “Dual Acting FAb” or “DAF” comprising an antigen binding site that binds to Tau as well as another, different antigen (see, US 2008/0069820, for example).

7. Antibody Variants

In certain embodiments, amino acid sequence variants of the antibodies provided herein are contemplated. For example, it may be desirable to improve the binding affinity and/or other biological properties of the antibody. Amino acid sequence variants of an antibody may be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody, or by peptide synthesis. Such modifications include, for example, deletions from, and/or insertions into and/or substitutions of residues within the amino acid sequences of the antibody. Any combination of deletion, insertion, and substitution can be made to arrive at the final construct, provided that the final construct possesses the desired characteristics, e.g., antigen-binding.

a) Substitution, Insertion, and Deletion Variants

In certain embodiments, antibody variants having one or more amino acid substitutions are provided. Sites of interest for substitutional mutagenesis include the HVRs and FRs. Conservative substitutions are shown in Table 1 under the heading of “preferred substitutions.” More substantial changes are provided in Table 1 under the heading of “exemplary substitutions,” and as further described below in reference to amino acid side chain classes. Amino acid substitutions may be introduced into an antibody of interest and the products screened for a desired activity, e.g., retained/improved antigen binding, decreased immunogenicity, or improved ADCC or CDC.

TABLE 1 Original Exemplary Preferred Residue Substitutions Substitutions Ala (A) Val; Leu; Ile Val Arg (R) Lys; Gln; Asn Lys Asn (N) Gln; His; Asp, Lys; Arg Gln Asp (D) Glu; Asn Glu Cys (C) Ser; Ala Ser Gln (Q) Asn; Glu Asn Glu (E) Asp; Gln Asp Gly (G) Ala Ala His (H) Asn; Gln; Lys; Arg Arg Ile (I) Leu; Val; Met; Ala; Phe; Norleucine Leu Leu (L) Norleucine; Ile; Val; Met; Ala; Phe Ile Lys (K) Arg; Gln; Asn Arg Met (M) Leu; Phe; Ile Leu Phe (F) Trp; Leu; Val; Ile; Ala; Tyr Tyr Pro (P) Ala Ala Ser (S) Thr Thr Thr (T) Val; Ser Ser Trp (W) Tyr; Phe Tyr Tyr (Y) Trp; Phe; Thr; Ser Phe Val (V) Ile; Leu; Met; Phe; Ala; Norleucine Leu Amino acids may be grouped according to common side-chain properties:

-   -   (1) hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile;     -   (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;     -   (3) acidic: Asp, Glu;     -   (4) basic: His, Lys, Arg;     -   (5) residues that influence chain orientation: Gly, Pro;     -   (6) aromatic: Trp, Tyr, Phe.

Non-conservative substitutions will entail exchanging a member of one of these classes for another class.

One type of substitutional variant involves substituting one or more hypervariable region residues of a parent antibody (e.g. a humanized or human antibody). Generally, the resulting variant(s) selected for further study will have modifications (e.g., improvements) in certain biological properties (e.g., increased affinity, reduced immunogenicity) relative to the parent antibody and/or will have substantially retained certain biological properties of the parent antibody. An exemplary substitutional variant is an affinity matured antibody, which may be conveniently generated, e.g., using phage display-based affinity maturation techniques such as those described herein. Briefly, one or more HVR residues are mutated and the variant antibodies displayed on phage and screened for a particular biological activity (e.g. binding affinity).

Alterations (e.g., substitutions) may be made in HVRs, e.g., to improve antibody affinity. Such alterations may be made in HVR “hotspots,” i.e., residues encoded by codons that undergo mutation at high frequency during the somatic maturation process (see, e.g., Chowdhury, Methods Mol. Biol. 207:179-196 (2008)), and/or residues that contact antigen, with the resulting variant VH or VL being tested for binding affinity. Affinity maturation by constructing and reselecting from secondary libraries has been described, e.g., in Hoogenboom et al. in Methods in Molecular Biology 178:1-37 (O'Brien et al., ed., Human Press, Totowa, N.J., (2001).) In some embodiments of affinity maturation, diversity is introduced into the variable genes chosen for maturation by any of a variety of methods (e.g., error-prone PCR, chain shuffling, or oligonucleotide-directed mutagenesis). A secondary library is then created. The library is then screened to identify any antibody variants with the desired affinity. Another method to introduce diversity involves HVR-directed approaches, in which several HVR residues (e.g., 4-6 residues at a time) are randomized. HVR residues involved in antigen binding may be specifically identified, e.g., using alanine scanning mutagenesis or modeling. CDR-H3 and CDR-L3 in particular are often targeted.

In certain embodiments, substitutions, insertions, or deletions may occur within one or more HVRs so long as such alterations do not substantially reduce the ability of the antibody to bind antigen. For example, conservative alterations (e.g., conservative substitutions as provided herein) that do not substantially reduce binding affinity may be made in HVRs. Such alterations may, for example, be outside of antigen contacting residues in the HVRs. In certain embodiments of the variant VH and VL sequences provided above, each HVR either is unaltered, or contains no more than one, two or three amino acid substitutions.

A useful method for identification of residues or regions of an antibody that may be targeted for mutagenesis is called “alanine scanning mutagenesis” as described by Cunningham and Wells (1989) Science, 244:1081-1085. In this method, a residue or group of target residues (e.g., charged residues such as arg, asp, his, lys, and glu) are identified and replaced by a neutral or negatively charged amino acid (e.g., alanine or polyalanine) to determine whether the interaction of the antibody with antigen is affected. Further substitutions may be introduced at the amino acid locations demonstrating functional sensitivity to the initial substitutions. Alternatively, or additionally, a crystal structure of an antigen-antibody complex to identify contact points between the antibody and antigen. Such contact residues and neighboring residues may be targeted or eliminated as candidates for substitution. Variants may be screened to determine whether they contain the desired properties.

Amino acid sequence insertions include amino- and/or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing a hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include an antibody with an N-terminal methionyl residue. Other insertional variants of the antibody molecule include the fusion to the N- or C-terminus of the antibody to an enzyme (e.g. for ADEPT) or a polypeptide which increases the serum half-life of the antibody.

b) Glycosylation Variants

In certain embodiments, an antibody provided herein is altered to increase or decrease the extent to which the antibody is glycosylated. Addition or deletion of glycosylation sites to an antibody may be conveniently accomplished by altering the amino acid sequence such that one or more glycosylation sites is created or removed.

Where the antibody comprises an Fc region, the carbohydrate attached thereto may be altered. Native antibodies produced by mammalian cells typically comprise a branched, biantennary oligosaccharide that is generally attached by an N-linkage to Asn297 of the CH2 domain of the Fc region. See, e.g., Wright et al. TIBTECH 15:26-32 (1997). The oligosaccharide may include various carbohydrates, e.g., mannose, N-acetyl glucosamine (GlcNAc), galactose, and sialic acid, as well as a fucose attached to a GlcNAc in the “stem” of the biantennary oligosaccharide structure. In some embodiments, modifications of the oligosaccharide in an antibody of the invention may be made in order to create antibody variants with certain improved properties.

In some embodiments, antibody variants are provided having a carbohydrate structure that lacks fucose attached (directly or indirectly) to an Fc region. For example, the amount of fucose in such antibody may be from 1% to 80%, from 1% to 65%, from 5% to 65% or from 20% to 40%. The amount of fucose is determined by calculating the average amount of fucose within the sugar chain at Asn297, relative to the sum of all glycostructures attached to Asn 297 (e. g. complex, hybrid and high mannose structures) as measured by MALDI-TOF mass spectrometry, as described in WO 2008/077546, for example. Asn297 refers to the asparagine residue located at about position 297 in the Fc region (Eu numbering of Fc region residues); however, Asn297 may also be located about ±3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300, due to minor sequence variations in antibodies. Such fucosylation variants may have improved ADCC function. See, e.g., US Patent Publication Nos. US 2003/0157108 (Presta, L.); US 2004/0093621 (Kyowa Hakko Kogyo Co., Ltd). Examples of publications related to “defucosylated” or “fucose-deficient” antibody variants include: US 2003/0157108; WO 2000/61739; WO 2001/29246; US 2003/0115614; US 2002/0164328; US 2004/0093621; US 2004/0132140; US 2004/0110704; US 2004/0110282; US 2004/0109865; WO 2003/085119; WO 2003/084570; WO 2005/035586; WO 2005/035778; WO2005/053742; WO2002/031140; Okazaki et al. J. Mol. Biol. 336:1239-1249 (2004); Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004). Examples of cell lines capable of producing defucosylated antibodies include Lec13 CHO cells deficient in protein fucosylation (Ripka et al. Arch. Biochem. Biophys. 249:533-545 (1986); US Pat Appl No US 2003/0157108 A1, Presta, L; and WO 2004/056312 A1, Adams et al., especially at Example 11), and knockout cell lines, such as alpha-1,6-fucosyltransferase gene, FUT8, knockout CHO cells (see, e.g., Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004); Kanda, Y. et al., Biotechnol. Bioeng., 94(4):680-688 (2006); and WO2003/085107).

Antibodies variants are further provided with bisected oligosaccharides, e.g., in which a biantennary oligosaccharide attached to the Fc region of the antibody is bisected by GlcNAc. Such antibody variants may have reduced fucosylation and/or improved ADCC function. Examples of such antibody variants are described, e.g., in WO 2003/011878 (Jean-Mairet et al.); U.S. Pat. No. 6,602,684 (Umana et al.); and US 2005/0123546 (Umana et al.). Antibody variants with at least one galactose residue in the oligosaccharide attached to the Fc region are also provided. Such antibody variants may have improved CDC function. Such antibody variants are described, e.g., in WO 1997/30087 (Patel et al.); WO 1998/58964 (Raju, S.); and WO 1999/22764 (Raju, S.).

c) Fc Region Variants

In certain embodiments, one or more amino acid modifications may be introduced into the Fc region of an antibody provided herein, thereby generating an Fc region variant. The Fc region variant may comprise a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3 or IgG4 Fc region) comprising an amino acid modification (e.g. a substitution) at one or more amino acid positions.

In certain embodiments, the invention contemplates an antibody variant that possesses some but not all effector functions, which make it a desirable candidate for applications in which the half life of the antibody in vivo is important yet certain effector functions (such as complement and ADCC) are unnecessary or deleterious. In vitro and/or in vivo cytotoxicity assays can be conducted to confirm the reduction/depletion of CDC and/or ADCC activities. For example, Fc receptor (FcR) binding assays can be conducted to ensure that the antibody lacks FcγR binding (hence likely lacking ADCC activity), but retains FcRn binding ability. The primary cells for mediating ADCC, NK cells, express FcγRIII only, whereas monocytes express FcγRI, FcγRII and FcγRIII. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991). Non-limiting examples of in vitro assays to assess ADCC activity of a molecule of interest is described in U.S. Pat. No. 5,500,362 (see, e.g. Hellstrom, I. et al. Proc. Nat'l Acad. Sci. USA 83:7059-7063 (1986)) and Hellstrom, I et al., Proc. Nat'l Acad. Sci. USA 82:1499-1502 (1985); 5,821,337 (see Bruggemann, M. et al., J. Exp. Med. 166:1351-1361 (1987)). Alternatively, non-radioactive assays methods may be employed (see, for example, ACTI™ non-radioactive cytotoxicity assay for flow cytometry (CellTechnology, Inc. Mountain View, Calif.; and CytoTox 96® non-radioactive cytotoxicity assay (Promega, Madison, Wis.). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and Natural Killer (NK) cells. Alternatively, or additionally, ADCC activity of the molecule of interest may be assessed in vivo, e.g., in a animal model such as that disclosed in Clynes et al. Proc. Nat'l Acad. Sci. USA 95:652-656 (1998). C1q binding assays may also be carried out to confirm that the antibody is unable to bind C1q and hence lacks CDC activity. See, e.g., C1q and C3c binding ELISA in WO 2006/029879 and WO 2005/100402. To assess complement activation, a CDC assay may be performed (see, for example, Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996); Cragg, M. S. et al., Blood 101:1045-1052 (2003); and Cragg, M. S. and M. J. Glennie, Blood 103:2738-2743 (2004)). FcRn binding and in vivo clearance/half life determinations can also be performed using methods known in the art (see, e.g., Petkova, S. B. et al., Int'l. Immunol. 18(12):1759-1769 (2006)).

Antibodies with reduced effector function include those with substitution of one or more of Fc region residues 238, 265, 269, 270, 297, 327 and 329 (U.S. Pat. No. 6,737,056). Such Fc mutants include Fc mutants with substitutions at two or more of amino acid positions 265, 269, 270, 297 and 327, including the so-called “DANA” Fc mutant with substitution of residues 265 and 297 to alanine (U.S. Pat. No. 7,332,581).

Certain antibody variants with improved or diminished binding to FcRs are described. (See, e.g., U.S. Pat. No. 6,737,056; WO 2004/056312, and Shields et al., J. Biol. Chem. 9(2): 6591-6604 (2001).)

In certain embodiments, an antibody variant comprises an Fc region with one or more amino acid substitutions which improve ADCC, e.g., substitutions at positions 298, 333, and/or 334 of the Fc region (EU numbering of residues).

In some embodiments, alterations are made in the Fc region that result in altered (i.e., either improved or diminished) C1q binding and/or Complement Dependent Cytotoxicity (CDC), e.g., as described in U.S. Pat. No. 6,194,551, WO 99/51642, and Idusogie et al. J. Immunol. 164: 4178-4184 (2000).

Antibodies with increased half lives and improved binding to the neonatal Fc receptor (FcRn), which is responsible for the transfer of maternal IgGs to the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)), are described in US2005/0014934A1 (Hinton et al.). Those antibodies comprise an Fc region with one or more substitutions therein which improve binding of the Fc region to FcRn. Such Fc variants include those with substitutions at one or more of Fc region residues: 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424, 428, or 434, e.g., substitution of Fc region residue 434 (U.S. Pat. No. 7,371,826).

See also Duncan & Winter, Nature 322:738-40 (1988); U.S. Pat. Nos. 5,648,260; 5,624,821; and WO 94/29351 concerning other examples of Fc region variants.

d) Cysteine Engineered Antibody Variants

In certain embodiments, it may be desirable to create cysteine engineered antibodies, e.g., “thioMAbs,” in which one or more residues of an antibody are substituted with cysteine residues. In particular embodiments, the substituted residues occur at accessible sites of the antibody. By substituting those residues with cysteine, reactive thiol groups are thereby positioned at accessible sites of the antibody and may be used to conjugate the antibody to other moieties, such as drug moieties or linker-drug moieties, to create an immunoconjugate, as described further herein. In certain embodiments, any one or more of the following residues may be substituted with cysteine: V205 (Kabat numbering) of the light chain; A118 (EU numbering) of the heavy chain; and 5400 (EU numbering) of the heavy chain Fc region. Cysteine engineered antibodies may be generated as described, e.g., in U.S. Pat. No. 7,521,541.

e) Antibody Derivatives

In certain embodiments, an antibody provided herein may be further modified to contain additional nonproteinaceous moieties that are known in the art and readily available. The moieties suitable for derivatization of the antibody include but are not limited to water soluble polymers. Non-limiting examples of water soluble polymers include, but are not limited to, polyethylene glycol (PEG), copolymers of ethylene glycol/propylene glycol, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene/maleic anhydride copolymer, polyaminoacids (either homopolymers or random copolymers), and dextran or poly(n-vinyl pyrrolidone)polyethylene glycol, propropylene glycol homopolymers, prolypropylene oxide/ethylene oxide co-polymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may have advantages in manufacturing due to its stability in water. The polymer may be of any molecular weight, and may be branched or unbranched. The number of polymers attached to the antibody may vary, and if more than one polymer are attached, they can be the same or different molecules. In general, the number and/or type of polymers used for derivatization can be determined based on considerations including, but not limited to, the particular properties or functions of the antibody to be improved, whether the antibody derivative will be used in a therapy under defined conditions, etc.

In another embodiment, conjugates of an antibody and nonproteinaceous moiety that may be selectively heated by exposure to radiation are provided. In some embodiments, the nonproteinaceous moiety is a carbon nanotube (Kam et al., Proc. Natl. Acad. Sci. USA 102: 11600-11605 (2005)). The radiation may be of any wavelength, and includes, but is not limited to, wavelengths that do not harm ordinary cells, but which heat the nonproteinaceous moiety to a temperature at which cells proximal to the antibody-nonproteinaceous moiety are killed.

B. Recombinant Methods and Compositions

Antibodies may be produced using recombinant methods and compositions, e.g., as described in U.S. Pat. No. 4,816,567. In some embodiments, isolated nucleic acid encoding an anti-Tau antibody described herein is provided. Such nucleic acid may encode an amino acid sequence comprising the VL and/or an amino acid sequence comprising the VH of the antibody (e.g., the light and/or heavy chains of the antibody). In a further embodiment, one or more vectors (e.g., expression vectors) comprising such nucleic acid are provided. In a further embodiment, a host cell comprising such nucleic acid is provided. In one such embodiment, a host cell comprises (e.g., has been transformed with): (1) a vector comprising a nucleic acid that encodes an amino acid sequence comprising the VL of the antibody and an amino acid sequence comprising the VH of the antibody, or (2) a first vector comprising a nucleic acid that encodes an amino acid sequence comprising the VL of the antibody and a second vector comprising a nucleic acid that encodes an amino acid sequence comprising the VH of the antibody. In some embodiments, the host cell is eukaryotic, e.g. a Chinese Hamster Ovary (CHO) cell or lymphoid cell (e.g., Y0, NS0, Sp20 cell). In some embodiments, a method of making an anti-Tau antibody is provided, wherein the method comprises culturing a host cell comprising a nucleic acid encoding the antibody, as provided above, under conditions suitable for expression of the antibody, and optionally recovering the antibody from the host cell (or host cell culture medium).

For recombinant production of an anti-Tau antibody, nucleic acid encoding an antibody, e.g., as described above, is isolated and inserted into one or more vectors for further cloning and/or expression in a host cell. Such nucleic acid may be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of the antibody).

Suitable host cells for cloning or expression of antibody-encoding vectors include prokaryotic or eukaryotic cells described herein. For example, antibodies may be produced in bacteria, in particular when glycosylation and Fc effector function are not needed. For expression of antibody fragments and polypeptides in bacteria, see, e.g., U.S. Pat. Nos. 5,648,237, 5,789,199, and 5,840,523. (See also Charlton, Methods in Molecular Biology, Vol. 248 (B. K. C. Lo, ed., Humana Press, Totowa, N.J., 2003), pp. 245-254, describing expression of antibody fragments in E. coli.) After expression, the antibody may be isolated from the bacterial cell paste in a soluble fraction and can be further purified.

In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for antibody-encoding vectors, including fungi and yeast strains whose glycosylation pathways have been “humanized,” resulting in the production of an antibody with a partially or fully human glycosylation pattern. See Gerngross, Nat. Biotech. 22:1409-1414 (2004), and Li et al., Nat. Biotech. 24:210-215 (2006).

Suitable host cells for the expression of glycosylated antibody are also derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant and insect cells. Numerous baculoviral strains have been identified which may be used in conjunction with insect cells, particularly for transfection of Spodoptera frugiperda cells.

Plant cell cultures can also be utilized as hosts. See, e.g., U.S. Pat. Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (describing PLANTIBODIES™ technology for producing antibodies in transgenic plants).

Vertebrate cells may also be used as hosts. For example, mammalian cell lines that are adapted to grow in suspension may be useful. Other examples of useful mammalian host cell lines are monkey kidney CV1 line transformed by SV40 (COS-7); human embryonic kidney line (293 or 293 cells as described, e.g., in Graham et al., J. Gen Virol. 36:59 (1977)); baby hamster kidney cells (BHK); mouse sertoli cells (TM4 cells as described, e.g., in Mather, Biol. Reprod. 23:243-251 (1980)); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical carcinoma cells (HELA); canine kidney cells (MDCK; buffalo rat liver cells (BRL 3A); human lung cells (W138); human liver cells (Hep G2); mouse mammary tumor (MMT 060562); TRI cells, as described, e.g., in Mather et al., Annals N.Y. Acad. Sci. 383:44-68 (1982); MRC 5 cells; and FS4 cells. Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR⁻ CHO cells (Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)); and myeloma cell lines such as Y0, NS0 and Sp2/0. For a review of certain mammalian host cell lines suitable for antibody production, see, e.g., Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (B. K. C. Lo, ed., Humana Press, Totowa, N.J.), pp. 255-268 (2003).

C. Assays

Anti-Tau antibodies provided herein may be identified, screened for, or characterized for their physical/chemical properties and/or biological activities by various assays known in the art.

1. Binding Assays and Other Assays

In one aspect, an antibody of the invention is tested for its antigen binding activity, e.g., by known methods such as ELISA, Western blot, etc.

In another aspect, competition assays may be used to identify an antibody that competes with an antibody described herein for binding to Tau. In certain embodiments, such a competing antibody binds to the same epitope (e.g., a linear or a conformational epitope) that is bound by 37D3-H9, hu37D3-H9.v28.A4, hu37D3-H9.v76, hu37D3-H9.v83, or hu37D3-H9.v93. Detailed exemplary methods for mapping an epitope to which an antibody binds are provided in Morris (1996) “Epitope Mapping Protocols,” in Methods in Molecular Biology vol. 66 (Humana Press, Totowa, N.J.).

In an exemplary competition assay, immobilized Tau (such as monomeric Tau) is incubated in a solution comprising a first labeled antibody that binds to Tau (e.g., any antibody described herein, such as hu37D3-H9.v28.A4) and a second unlabeled antibody that is being tested for its ability to compete with the first antibody for binding to Tau. The second antibody may be present in a hybridoma supernatant. As a control, immobilized Tau is incubated in a solution comprising the first labeled antibody but not the second unlabeled antibody. After incubation under conditions permissive for binding of the first antibody to Tau, excess unbound antibody is removed, and the amount of label associated with immobilized Tau is measured. If the amount of label associated with immobilized Tau is substantially reduced in the test sample relative to the control sample, then that indicates that the second antibody is competing with the first antibody for binding to Tau. See Harlow and Lane (1988) Antibodies: A Laboratory Manual ch. 14 (Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.).

2. Activity Assays

In one aspect, assays are provided for identifying anti-Tau (e.g., pan-Tau) antibodies thereof having biological activity. Biological activity may include, e.g., binding of such antibodies to multiple forms of Tau (e.g., monomeric Tau, oligomeric Tau, non-phosphorylated Tau, and phosphorylated Tau) and reducing the level of Tau protein (e.g., total Tau, total soluble Tau, soluble non-phosphorylated Tau, soluble phosphorylated Tau, total insoluble Tau, insoluble non-phosphorylated Tau, insoluble phosphorylated Tau, hyperphosphorylated Tau, or paired helical filaments containing hyperphosphorylated Tau, in the brain, e.g., in the brain cortex and/or hippocampus). Antibodies having such biological activity in vivo and/or in vitro are also provided.

In certain embodiments, an antibody of the invention is tested for such biological activity. For example, an animal model of tauopathy, such as a Tau transgenic mice (e.g., P301L), can be used to detect binding of anti-Tau antibodies to brain sections, and for example, to neurofibrillary tangles in the brains of the transgenic mice. Further, an animal model of tauopathy, such as a Tau transgenic mice (e.g., P301L), can be treated with anti-Tau antibodies and experimental techniques known in the art can be used to assess whether such treatment reduces the level of Tau protein (e.g., total Tau, total soluble Tau, soluble phosphorylated Tau, soluble non-phosphorylated Tau, total insoluble Tau, insoluble phosphorylated Tau, insoluble non-phosphorylated Tau, hyperphosphorylated Tau, or paired helical filaments containing hyperphosphorylated Tau) in the mouse brain (e.g., in the brain cortex and/or hippocampus).

D. Immunoconjugates

The invention also provides immunoconjugates comprising an anti-Tau antibody herein conjugated to one or more other therapeutic agents or radioactive isotopes.

In another embodiment, an immunoconjugate comprises an antibody as described herein conjugated to a radioactive atom to form a radioconjugate. A variety of radioactive isotopes are available for the production of radioconjugates. Examples include At²¹¹, I³¹¹, I¹²⁵, Y⁹⁰, Re¹⁸⁶, Re¹⁸⁸, Sm¹⁵³, Bi²¹², P³², Pb²¹² and radioactive isotopes of Lu. When the radioconjugate is used for detection, it may comprise a radioactive atom for scintigraphic studies, for example tc99m or I123, or a spin label for nuclear magnetic resonance (NMR) imaging (also known as magnetic resonance imaging, mri), such as iodine-123 again, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese or iron.

Conjugates of an antibody may be made using a variety of bifunctional protein coupling agents such as N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP), succinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCl), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate), and bis-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin can be prepared as described in Vitetta et al., Science 238:1098 (1987). Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugation of radionucleotide to the antibody. See WO94/11026. The linker may be a “cleavable linker” facilitating release of a cytotoxic drug in the cell. For example, an acid-labile linker, peptidase-sensitive linker, photolabile linker, dimethyl linker or disulfide-containing linker (Chari et al., Cancer Res. 52:127-131 (1992); U.S. Pat. No. 5,208,020) may be used.

The immunuoconjugates or ADCs herein expressly contemplate, but are not limited to such conjugates prepared with cross-linker reagents including, but not limited to, BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, and sulfo-SMPB, and SVSB (succinimidyl-(4-vinyl sulfone)benzoate) which are commercially available (e.g., from Pierce Biotechnology, Inc., Rockford, Ill., U.S.A).

E. Methods and Compositions for Diagnostics and Detection

In certain embodiments, any of the anti-Tau antibodies provided herein is useful for detecting the presence of Tau in a biological sample. The term “detecting” as used herein encompasses quantitative or qualitative detection. In certain embodiments, a biological sample comprises a cell or tissue, such as cerebrospinal fluid, a cell or tissue of the brain (e.g., brain cortex or hippocampus), or blood. In some embodiments, a biological sample is cerebrospinal fluid.

In some embodiments, an anti-Tau antibody for use in a method of diagnosis or detection is provided. In a further aspect, a method of detecting the presence of Tau in a biological sample is provided. In certain embodiments, the method comprises contacting the biological sample with an anti-Tau antibody as described herein under conditions permissive for binding of the anti-Tau antibody to Tau, and detecting whether a complex is formed between the anti-Tau antibody and Tau. Such method may be an in vitro or in vivo method. Further, the complex formed between the anti-Tau antibody and Tau in a test biological sample can be compared to the complex formed in a control biological sample (e.g., a biological sample from a healthy subject or subjects). The amount of the complex formed between the anti-Tau antibody and Tau in a test biological sample can also be quantified and compared to the amount of the complex formed in a control biological sample (e.g., a biological sample from a healthy subject or subjects) or to the average amount of the complex known to be formed in healthy subjects.

In some embodiments, an anti-Tau antibody is used to select subjects eligible for therapy with an anti-Tau antibody, e.g. where Tau is a biomarker for selection of patients. For example, in some embodiments, an anti-Tau (e.g., pan-Tau) antibody is used to detect whether the subject has a Tau protein disease or disorder, or whether the subject is at high risk (or predisposed to) a Tau protein disease or disorder.

Exemplary diseases or disorders that may be diagnosed using an antibody of the invention include Tau protein associated diseases or disorders, and diseases or disorders caused by or associated with the formation of neurofibrillary tangles or neuropil threads. In some embodiments, diseases or disorders that may be diagnosed using an antibody of the invention include Tau protein associated diseases or disorders that are manifested in an impairment or loss of cognitive functions including reasoning, situational judgement, memory capacity, learning, and/or special navigation. In particular, diseases or disorders that may be diagnosed using an antibody of the invention include tauopathies such as neurodegenerative tauopathies. Exemplary diseases or disorders that may be diagnosed using an antibody of the invention include, but are not limited to, Alzheimer's Disease, Creutzfeldt-Jacob disease, Dementia pugilistica, Down's Syndrome, Gerstmann-Sträussler-Scheinker disease, inclusion-body myositis, prion protein cerebral amyloid angiopathy, traumatic brain injury, amyotrophic lateral sclerosis/parkinsonism-dementia complex of Guam, Non-Guamanian motor neuron disease with neurofibrillary tangles, argyrophilic grain dementia, corticobasal degeneration, diffuse neurofibrillary tangles with calcification, frontotetemporal dementia, frontotemporal dementia with parkinsonism linked to chromosome 17, Hallevorden-Spatz disease, multiple system atrophy, Niemann-Pick disease type C, Pallido-ponto-nigral degeneration, Pick's disease, progressive subcortical gliosis, progressive supranuclear palsy, Subacute sclerosing panencephalitis, Tangle only dementia, Postencephalitic Parkinsonism, and Myotonic dystrophy. Further nonlimiting exemplary diseases and disorders that may be diagnosed using an antibody of the invention include PART (primary age-related Tauopathy), tangle predominant dementia, subacute sclerosis panencephalopathy, chronic traumatic encephalopathy (CTE), white matter tauopathy with globular glial inclusions, Lewy body dementia (LBD), mild cognitive impairment (MCI), glaucoma, familial British dementia, familiar Danish dementia, Guadeloupean Parkinsonism, neurodegeneration with brain iron accumulation, SLC9A6-related mental retardation, multiple sclerosis, HIV-related dementia, senile cardiac amyloidosis, and Huntington's disease. In some embodiments, a disorder that may be diagnosed using an antibody of the invention is Alzheimer's Disease (AD).

In certain embodiments, labeled anti-Tau antibodies are provided. Labels include, but are not limited to, labels or moieties that are detected directly (such as fluorescent, chromophoric, electron-dense, chemiluminescent, and radioactive labels), as well as moieties, such as enzymes or ligands, that are detected indirectly, e.g., through an enzymatic reaction or molecular interaction. Exemplary labels include, but are not limited to, the radioisotopes ³²P, ¹⁴C, ¹²⁵I, ³H, and ¹³¹I, fluorophores such as rare earth chelates or fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone, luceriferases, e.g., firefly luciferase and bacterial luciferase (U.S. Pat. No. 4,737,456), luciferin, 2,3-dihydrophthalazinediones, horseradish peroxidase (HRP), alkaline phosphatase, β-galactosidase, glucoamylase, lysozyme, saccharide oxidases, e.g., glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase, heterocyclic oxidases such as uricase and xanthine oxidase, coupled with an enzyme that employs hydrogen peroxide to oxidize a dye precursor such as HRP, lactoperoxidase, or microperoxidase, biotin/avidin, spin labels, bacteriophage labels, stable free radicals, and the like.

F. Pharmaceutical Formulations

Pharmaceutical formulations of an anti-Tau antibody as described herein are prepared by mixing such antibody having the desired degree of purity with one or more optional pharmaceutically acceptable carriers, diluents, and/or excipients (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)), in the form of lyophilized formulations or aqueous solutions. Pharmaceutically acceptable carriers, diluents, and excipients are generally nontoxic to recipients at the dosages and concentrations employed, and include, but are not limited to: sterile water, buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g. Zn-protein complexes); and/or non-ionic surfactants such as polyethylene glycol (PEG). Exemplary pharmaceutically acceptable carriers herein further include insterstitial drug dispersion agents such as soluble neutral-active hyaluronidase glycoproteins (sHASEGP), for example, human soluble PH-20 hyaluronidase glycoproteins, such as rHuPH20 (HYLENEX®, Baxter International, Inc.). Certain exemplary sHASEGPs and methods of use, including rHuPH20, are described in US Patent Publication Nos. 2005/0260186 and 2006/0104968. In one aspect, a sHASEGP is combined with one or more additional glycosaminoglycanases such as chondroitinases.

Exemplary lyophilized antibody formulations are described in U.S. Pat. No. 6,267,958. Aqueous antibody formulations include those described in U.S. Pat. No. 6,171,586 and WO2006/044908, the latter formulations including a histidine-acetate buffer.

The formulation herein may also contain more than one active ingredients as necessary for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other. Such active ingredients are suitably present in combination in amounts that are effective for the purpose intended.

Active ingredients may be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsules and poly-(methylmethacylate) microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules) or in macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

Sustained-release preparations may be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g. films, or microcapsules.

The formulations to be used for in vivo administration are generally sterile. Sterility may be readily accomplished, e.g., by filtration through sterile filtration membranes.

G. Therapeutic Methods and Compositions

Any of the anti-Tau antibodies provided herein may be used in therapeutic methods.

In one aspect, an anti-Tau antibody for use as a medicament is provided. In further aspects, an anti-Tau antibody for use in treating a Tau protein associated disease or disorder is provided. In some embodiments, an anti-Tau antibody for use in treating diseases or disorders caused by or associated with the formation of neurofibrillary tangles or neuropil threads is provided. In particular embodiments, an anti-Tau antibody for use in treating a tauopathy such as a neurodegenerative tauopathy is provided. Exemplary Tau protein associated diseases or disorders that can be treated that can be treated with anti-tau antibodies include, without limitation, Alzheimer's Disease, amyotrophic lateral sclerosis, Parkinson's disease, Creutzfeldt-Jacob disease, Dementia pugilistica, Down's Syndrome, Gerstmann-Straussler-Scheinker disease, inclusion-body myositis, prion protein cerebral amyloid angiopathy, traumatic brain injury, amyotrophic lateral sclerosis/parkinsonism-dementia complex of Guam, Non-Guamanian motor neuron disease with neurofibrillary tangles, argyrophilic grain dementia, corticobasal degeneration, diffuse neurofibrillary tangles with calcification, frontotetemporal dementia, frontotemporal dementia with parkinsonism linked to chromosome 17, Hallevorden-Spatz disease, multiple system atrophy, Niemann-Pick disease type C, Pallido-ponto-nigral degeneration, Pick's disease, progressive subcortical gliosis, progressive supranuclear palsy, Subacute sclerosing panencephalitis, Tangle only dementia, Postencephalitic Parkinsonism, and Myotonic dystrophy. Further exemplary Tau protein associated diseases or disorders that can be treated that can be treated with anti-tau antibodies include, without limitation, PART (primary age-related Tauopathy), tangle predominant dementia, subacute sclerosis panencephalopathy, chronic traumatic encephalopathy (CTE), white matter tauopathy with globular glial inclusions, Lewy body dementia (LBD), mild cognitive impairment (MCI), glaucoma, familial British dementia, familiar Danish dementia, Guadeloupean Parkinsonism, neurodegeneration with brain iron accumulation, SLC9A6-related mental retardation, multiple sclerosis, HIV-related dementia, senile cardiac amyloidosis, and Huntington's disease. In some embodiments, an anti-Tau antibody for use in treating Alzheimer's Disease (AD) is provided herein. In some embodiments, an anti-Tau antibody for use in treating moderate AD, mild to moderate AD, mild AD, early AD, or prodromal AD is provided herein. Further, Tau protein associated diseases or disorders that can be treated with an anti-Tau antibody include diseases or disorders that are manifested in an impairment or loss of a cognitive function such as reasoning, situational judgement, memory capacity, learning, and/or special navigation. In certain embodiments, an anti-Tau antibody for use in a method of treatment is provided. In certain embodiments, the invention provides an anti-Tau antibody for use in a method of treating an individual, having any one of the Tau associated diseases or disorders described above, comprising administering to the individual an effective amount of the anti-Tau antibody. In one such embodiment, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent, e.g., as described below.

In some embodiments, the antibody of the invention is used to treat an individual having an MMSE score of between 20 and 30, between 20 and 26, between 24 and 30, between 21 and 26, between 22 and 26, between 22 and 28, between 23 and 26, between 24 and 26, or between 25 and 26. In some embodiments, the patient has an MMSE score between 22 and 26. As used herein, an MMSE score between two numbers includes the numbers at each end of the range. For example, an MMSE score between 22 and 26 includes MMSE scores of 22 and 26.

In some embodiments, the antibodies of the invention are used to treat an individual who is ‘tau positive,’ e.g., a patient having brain tau deposits that are typical of Tau protein associated disorders, e.g., a patient having a positive Tau PET scan.

In further embodiments, the invention provides an anti-Tau antibody for use in reducing the levels of Tau protein (e.g., total Tau, total soluble Tau, soluble phosphorylated Tau, total insoluble Tau, insoluble phosphorylated Tau, hyperphosphorylated Tau, or paired helical filaments containing hyperphosphorylated Tau) in an individual. For example such reduction can occur in the brain (e.g., in the brain cortex and/or hippocampus). In some embodiments, the invention provides an anti-Tau antibody for use in reducing the levels of phosphorylated Tau. In some embodiments, the invention provides an anti-Tau antibody for use in reducing the levels of insoluble Tau (e.g., insoluble phosphorylated Tau). In some embodiments, the invention provides an anti-Tau antibody for use in reducing the levels of hyperphosphorylated Tau. In some embodiments, the invention provides an anti-Tau antibody for use in reducing the levels of paired helical filaments (e.g., paired helical filaments containing hyperphosphorylated Tau) in a brain tissue (e.g., in the brain cortex and/or hippocampus). In certain embodiments, the invention provides an anti-Tau antibody for use in a method of reducing the levels of Tau protein (e.g., total Tau, total soluble Tau, soluble phosphorylated Tau, total insoluble Tau, insoluble phosphorylated Tau, hyperphosphorylated Tau, or paired helical filaments containing hyperphosphorylated Tau) in the brain (e.g., in the brain cortex and/or hippocampus) in an individual comprising administering to the individual an effective amount of the anti-Tau antibody to reduce the levels of Tau protein. An “individual” according to any of the above embodiments is a mammal, preferably a human.

In some embodiments, the invention provides an anti-Tau antibody for use in modulating the levels of Tau protein (e.g., total Tau, total soluble Tau, soluble phosphorylated Tau, total insoluble Tau, insoluble phosphorylated Tau, hyperphosphorylated Tau, or paired helical filaments containing hyperphosphorylated Tau), for example, in the brain (e.g., in the brain cortex and/or hippocampus) of an individual.

In a further aspect, the invention provides for the use of an anti-Tau antibody in the manufacture or preparation of a medicament. In some embodiments, the medicament is for treatment of a Tau protein associated disease or disorder. The Tau protein associated disease or disorder can be a disease or disorders caused by or associated with the formation of neurofibrillary tangles or neuropil threads. In particular embodiments, the medicament is for treatment of a tauopathy such as a neurodegenerative tauopathy. In specific embodiments, the medicament is for treatment of diseases or disorders selected from the group consisting of: Alzheimer's Disease (AD), Creutzfeldt-Jacob disease, Dementia pugilistica, Down's Syndrome, Gerstmann-Sträussler-Scheinker disease, inclusion-body myositis, prion protein cerebral amyloid angiopathy, traumatic brain injury, amyotrophic lateral sclerosis/parkinsonism-dementia complex of Guam, Non-Guamanian motor neuron disease with neurofibrillary tangles, argyrophilic grain dementia, corticobasal degeneration, diffuse neurofibrillary tangles with calcification, frontotetemporal dementia, frontotemporal dementia with parkinsonism linked to chromosome 17, Hallevorden-Spatz disease, multiple system atrophy, Niemann-Pick disease type C, Pallido-ponto-nigral degeneration, Pick's disease, progressive subcortical gliosis, progressive supranuclear palsy, Subacute sclerosing panencephalitis, Tangle only dementia, Postencephalitic Parkinsonism, and Myotonic dystrophy. In some embodiments, the medicament is for treatment of diseases or disorders selected from PART (primary age-related Tauopathy), tangle predominant dementia, subacute sclerosis panencephalopathy, chronic traumatic encephalopathy (CTE), white matter tauopathy with globular glial inclusions, Lewy body dementia (LBD), mild cognitive impairment (MCI), glaucoma, familial British dementia, familiar Danish dementia, Guadeloupean Parkinsonism, neurodegeneration with brain iron accumulation, SLC9A6-related mental retardation, multiple sclerosis, HIV-related dementia, senile cardiac amyloidosis, and Huntington's disease. In some embodiments, the medicament is for treatment of AD. In particular embodiments, the medicament is for treatment of a Tau associated disease or disorder that is manifested in an impairment or loss of a cognitive function such as reasoning, situational judgement, memory capacity, learning, or special navigation. In a further embodiment, the medicament is for use in a method of treating one of the above-listed diseases (e.g., a tauopathy such as AD) comprising administering to an individual having such disease an effective amount of the medicament. In one such embodiment, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent, e.g., as described below.

In a further embodiment, the medicament is for reducing the levels of Tau protein (e.g., total Tau, total soluble Tau, soluble non-phorphorylated Tau, soluble phosphorylated Tau, total insoluble Tau, insoluble phosphorylated Tau, insoluble non-phorphorylated Tau, hyperphosphorylated Tau, or paired helical filaments containing hyperphosphorylated Tau). For example, such reducing of Tau protein can be observed in the brain (e.g., in the brain cortex and/or hippocampus) or in cerebrospinal fluid of an individual. In some embodiments, the medicament is for reducing the levels of paired helical filaments. In a further embodiment, the medicament is for use in a method of reducing the levels of Tau protein (e.g., total Tau, total soluble Tau, soluble phosphorylated Tau, total insoluble Tau, insoluble phosphorylated Tau, hyperphosphorylated Tau, or paired helical filaments containing hyperphosphorylated Tau) in an individual comprising administering to the individual an effective amount of the medicament to reducing the levels of Tau protein. An “individual” according to any of the above embodiments is a mammal, preferably, a human.

In a further aspect, the invention provides a method for treating a Tau protein associated disease or disorder. Tau protein associated disease or disorder that can be treated in accordance with the methods provided herein include diseases or disorders caused by or associated with the formation of neurofibrillary tangles or neuropil threads. In particular embodiments, the invention provides a method for treating a tauopathy such as a neurodegenerative tauopathy. In specific embodiments, the invention provides a method for treating a disease or disorder selected from the group consisting of: Alzheimer's Disease, Creutzfeldt-Jacob disease, Dementia pugilistica, Down's Syndrome, Gerstmann-Straussler-Scheinker disease, inclusion-body myositis, prion protein cerebral amyloid angiopathy, traumatic brain injury, amyotrophic lateral sclerosis/parkinsonism-dementia complex of Guam, Non-Guamanian motor neuron disease with neurofibrillary tangles, argyrophilic grain dementia, corticobasal degeneration, diffuse neurofibrillary tangles with calcification, frontotetemporal dementia, frontotemporal dementia with parkinsonism linked to chromosome 17, Hallevorden-Spatz disease, multiple system atrophy, Niemann-Pick disease type C, Pallido-ponto-nigral degeneration, Pick's disease, progressive subcortical gliosis, progressive supranuclear palsy, Subacute sclerosing panencephalitis, Tangle only dementia, Postencephalitic Parkinsonism, and Myotonic dystrophy. In some embodiments, the invention provides methods for treating diseases or disorders selected from PART (primary age-related Tauopathy), tangle predominant dementia, subacute sclerosis panencephalopathy, chronic traumatic encephalopathy (CTE), white matter tauopathy with globular glial inclusions, Lewy body dementia (LBD), mild cognitive impairment (MCI), glaucoma, familial British dementia, familiar Danish dementia, Guadeloupean Parkinsonism, neurodegeneration with brain iron accumulation, SLC9A6-related mental retardation, multiple sclerosis, HIV-related dementia, senile cardiac amyloidosis, and Huntington's disease. In some embodiments, the invention provides a method for treating Alzheimer's Disease (AD). In particular embodiments, the invention provides a method for treating a Tau protein associated disease or disorder that is manifested in an impairment or loss of a cognitive function such as reasoning, situational judgement, memory capacity, learning, or special navigation. In some embodiments, the method comprises administering to an individual, having any one of the diseases or disorders described above, an effective amount of an anti-Tau antibody. In one such embodiment, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent, e.g., as described below. In some embodiments, the method comprises administering to an individual having one of the diseases described herein an effective amount of an anti-Tau antibody. In one such embodiment, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent, as described below. An “individual” according to any of the above embodiments may be a human.

In a further aspect, the invention provides a method for reducing the levels of Tau protein (e.g., total Tau, total soluble Tau, soluble phosphorylated Tau, total insoluble Tau, insoluble phosphorylated Tau, hyperphosphorylated Tau, or paired helical filaments containing hyperphosphorylated Tau) in an individual. For example, such reducing of the levels of Tau protein can be observed in the brain (e.g., brain cortex and/or hippocampus) or cerebrospinal fluid of an individual. In some embodiments, the invention provides a method for reducing the levels of paired helical filaments. In some embodiments, the method comprises administering to the individual an effective amount of an anti-Tau antibody to reduce the levels of Tau protein. In some embodiments, an “individual” is a human.

In some aspects, the invention provides a method for alleviating one or more symptoms of a Tau protein associated disease or disorder; or an anti-Tau antibody or a medicament comprising anti-Tau antibody for alleviating one or more symptoms of a Tau protein associated disease or disorder (such as any of the diseases or disorders described herein, for example, AD). In some aspects, the invention provides a method for reducing the number of symptoms or the severity of one or more symptoms of a Tau protein associated disease or disorder; or an anti-Tau antibody or a medicament comprising anti-Tau antibody for reducing the number of symptoms or the severity of one or more symptoms of a Tau protein associated disease or disorder (such as any of the diseases or disorders described herein, for example, AD). In a particular embodiment, the symptom of a Tau protein associated disease or disorder is an impairment in cognition. In a specific embodiment, the symptom of a Tau protein associated disease or disorder is an impairment in learning and/or memory. In a specific embodiment, the symptom of a Tau protein associated disease or disorder is a long-term memory loss. In a specific embodiment, the symptom of a Tau protein associated disease or disorder is dementia. In some embodiments, the symptom of a Tau protein associated disease or disorder is confusion, irritability, aggression, mood swings, or a language impairment. In some embodiments, the symptom of a Tau protein associated disease or disorder is an impairment or loss of one or more cognitive functions such as reasoning, situational judgment, memory capacity, and/or learning. The methods provided herein comprise administration of an amount (e.g., therapeutically effective amount) of an anti-Tau antibody to an individual (e.g., who displays one or more symptoms of a Tau protein associated disease or disorder).

In specific aspects, the invention provides a method for retaining or increasing cognitive memory capacity, or for slowing down memory loss associated with a Tau protein associated disease or disorder; or an anti-Tau antibody or a medicament comprising anti-Tau antibody for retaining or increasing cognitive memory capacity or for slowing down memory loss associated with a Tau protein associated disease or disorder (such as any of the diseases or disorders described herein, for example, AD). The methods provided herein comprise administration of an amount (e.g., therapeutically effective amount) of an anti-Tau antibody to an individual (e.g., who displays one or more symptoms of memory loss or a decrease of memory capacity).

In some aspects, the invention provides a method for decreasing the rate of progression of a Tau protein associated disease or disorder; or an anti-Tau antibody or a medicament comprising anti-Tau antibody for decreasing the rate of progression of a Tau protein associated disease or disorder (such as any of the diseases or disorders described herein, for example, AD). The methods provided herein comprise administration of an amount (e.g., therapeutically effective amount) of an anti-Tau antibody to an individual (e.g., who displays one or more symptoms of a Tau protein associated disease or disorder).

In some aspects, the invention provides a method for preventing the development of a Tau protein associated disease or disorder; or an anti-Tau antibody or a medicament comprising anti-Tau antibody for preventing the development of a Tau protein associated disease or disorder (such as any of the diseases or disorders described herein, for example, AD). The methods provided herein comprise administration of an amount (e.g., therapeutically effective amount) of an anti-Tau antibody to an individual (e.g., who is at risk of a Tau protein associated disease or disorder).

In some aspects, the invention provides a method for delaying the development of a Tau protein associated disease or disorder; or an anti-Tau antibody or a medicament comprising anti-Tau antibody for delaying the development of a Tau protein associated disease or disorder (such as any of the diseases or disorders described herein, for example, AD). The methods provided herein comprise administration of an amount (e.g., therapeutically effective amount) of an anti-Tau antibody to an individual (e.g., who displays one or more symptoms of a Tau protein associated disease or disorder).

In a further aspect, the invention provides pharmaceutical formulations comprising any of the anti-Tau antibodies provided herein, e.g., for use in any of the above therapeutic methods. In some embodiments, a pharmaceutical formulation comprises any of the anti-Tau antibodies provided herein and a pharmaceutically acceptable carrier. In another embodiment, a pharmaceutical formulation comprises any of the anti-Tau antibodies provided herein and at least one additional therapeutic agent, e.g., as described below.

Antibodies of the invention can be used either alone or in combination with other agents in a therapy. For instance, an antibody of the invention may be co-administered with at least one additional therapeutic agent.

For example, the composition according to the invention may be administered in combination with other compositions comprising an additional therapeutic agent, such as a biologically active substance or compound such as, for example, a known compound used in the medication of tauopathies and/or of amyloidoses, a group of diseases and disorders associated with amyloid or amyloid-like protein such as the amyloid protein involved in Alzheimer's Disease.

Generally, the other biologically active compound may include neuron-transmission enhancers, psychotherapeutic drugs, acetylcholine esterase inhibitors, calcium-channel blockers, biogenic amines, benzodiazepine tranquillizers, acetylcholine synthesis, storage or release enhancers, acetylcholine postsynaptic receptor agonists, monoamine oxidase-A or -B inhibitors, N-methyl-D-aspartate glutamate receptor antagonists, non-steroidal anti-inflammatory drugs, antioxidants, serotonergic receptor antagonists, or other therapeutic agents. In particular, the biologically active agent or compound may comprise at least one compound selected from compounds against oxidative stress, anti-apoptotic compounds, metal chelators, inhibitors of DNA repair such as pirenzepine and metabolites, 3-amino-1-propanesulfonic acid (3APS), 1,3-propanedisulfonate (1,3PDS), secretase activators, beta- and gamma-secretase inhibitors, tau proteins, anti-Tau antibodies (including, but not limited to, antibodies disclosed in WO2012049570, WO2014028777, WO2014165271, WO2014100600, WO2015200806, U.S. Pat. Nos. 8,980,270, and 8,980,271), neurotransmitter, beta-sheet breakers, antiinflammatory molecules, “atypical antipsychotics” such as, for example clozapine, ziprasidone, risperidone, aripiprazole or olanzapine or cholinesterase inhibitors (ChEIs) such as tacrine, rivastigmine, donepezil, and/or galantamine and other drugs and nutritive supplements such as, for example, vitamin B 12, cysteine, a precursor of acetylcholine, lecithin, choline, Ginkgo biloba, acyetyl-L-carnitine, idebenone, propentofylline, or a xanthine derivative, together with a binding peptide according to the invention including antibodies, particularly monoclonal antibodies and active fragments thereof, and, optionally, a pharmaceutically acceptable carrier and/or a diluent and/or an excipient and instructions for the treatment of diseases.

In some embodiments, an antibody of the invention may be administered in combination with a neurological drug. Such neurological drugs include, but are not limited to, an antibody or other binding molecule (including, but not limited to a small molecule, a peptide, an aptamer, or other protein binder) that specifically binds to a target selected from: beta secretase, presenilin, amyloid precursor protein or portions thereof, amyloid beta peptide or oligomers or fibrils thereof, death receptor 6 (DR6), receptor for advanced glycation endproducts (RAGE), parkin, and huntingtin; an NMDA receptor antagonist (i.e., memantine), a monoamine depletor (i.e., tetrabenazine); an ergoloid mesylate; an anticholinergic antiparkinsonism agent (i.e., procyclidine, diphenhydramine, trihexylphenidyl, benztropine, biperiden and trihexyphenidyl); a dopaminergic antiparkinsonism agent (i.e., entacapone, selegiline, pramipexole, bromocriptine, rotigotine, selegiline, ropinirole, rasagiline, apomorphine, carbidopa, levodopa, pergolide, tolcapone and amantadine); a tetrabenazine; an anti-inflammatory (including, but not limited to, a nonsteroidal anti-inflammatory drug (i.e., indomethicin and other compounds listed above); a hormone (i.e., estrogen, progesterone and leuprolide); a vitamin (i.e., folate and nicotinamide); a dimebolin; a homotaurine (i.e., 3-aminopropanesulfonic acid; 3APS); a serotonin receptor activity modulator (i.e., xaliproden); an, an interferon, and a glucocorticoid or corticosteroid. The term “corticosteroid” includes, but is not limited to, fluticasone (including fluticasone propionate (FP)), beclometasone, budesonide, ciclesonide, mometasone, flunisolide, betamethasone and triamcinolone. “Inhalable corticosteroid” means a corticosteroid that is suitable for delivery by inhalation. Exemplary inhalable corticosteroids are fluticasone, beclomethasone dipropionate, budenoside, mometasone furoate, ciclesonide, flunisolide, and triamcinolone acetonide.

In some embodiments, one or more anti-amyloid beta (anti-Abeta) antibodies may be administered with an anti-Tau antibody provided herein. Non-limiting examples of such anti-Abeta antibodies include crenezumab, solanezumab, bapineuzumab, aducanumab, and BAN-2401 (Biogen, Eisai Co., Ltd.). In some embodiments, one or more beta-amyloid aggregation inhibitors may be administered with an anti-Tau antibody provided herein. Nonlimiting exemplary beta-amyloid aggregation inhibitors include ELND-005 (also referred to as AZD-103 or scyllo-inositol), tramiprosate, and PTI-80 (Exebryl-1®; ProteoTech). In some embodiments, one or more BACE inhibitors may be administered with an anti-Tau antibody provided herein. Non-limiting examples of such BACE inhibitors include E-2609 (Biogen, Eisai Co., Ltd.), AZD3293 (also known as LY3314814; AstraZeneca, Eli Lilly & Co.), MK-8931 (verubecestat), and JNJ-54861911 (Janssen, Shionogi Pharma). In some embodiments, one or more Tau inhibitors may be administered with an anti-Tau antibody provided herein. Non-limiting examples of such Tau inhibitors include methylthioninium, LMTX (also known as leuco-methylthioninium or Trx-0237; TauRx Therapeutics Ltd.), Rember™ (methylene blue or methylthioninium chloride [MTC]; Trx-0014; TauRx Therapeutics Ltd), PBT2 (Prana Biotechnology), and PTI-51-CH3 (TauPro™; ProteoTech). In some embodiments, one or more other anti-Tau antibodies may be administered with an anti-Tau antibody provided herein. Non-limiting examples of such other anti-Tau antibodies include BMS-986168 (Bristol-Myers Squibb) and C2N-8E12 (AbbVie, C2N Diagnostics, LLC). In some embodiments, a general misfolding inhibitor, such as NPT088 (NeuroPhage Pharmaceuticals), may be administered with an anti-Tau antibody provided herein.

In some embodiments, the composition according to the invention may comprise niacin or memantine together with a chimeric antibody or a humanized antibody according to the invention including antibodies, particularly monoclonal antibodies and active fragments thereof, and, optionally, a pharmaceutically acceptable carrier and/or a diluent and/or an excipient.

In some embodiments, compositions are provided that comprise “atypical antipsychotics” such as, for example clozapine, ziprasidone, risperidone, aripiprazole or olanzapine for the treatment of positive and negative psychotic symptoms including hallucinations, delusions, thought disorders (manifested by marked incoherence, derailment, tangentiality), and bizarre or disorganized behavior, as well as anhedonia, flattened affect, apathy, and social withdrawal, together with the chimeric antibody or the humanized antibody according to the invention or active fragments thereof, and, optionally, a pharmaceutically acceptable carrier and/or a diluent and/or an excipient.

Other compounds that can be suitably used in compositions in addition to chimeric antibody or humanized antibody according to the invention, are those disclosed, for example, in WO 2004/058258 (see especially pages 16 and 17) including therapeutic drug targets (page 36-39), alkanesulfonic acids and alkanolsulfuric acid (pages 39-51), cholinesterase inhibitors (pages 51-56), NMDA receptor antagonists (pages 56-58), estrogens (pages 58-59), non-steroidal anti-inflammatory drugs (pages 60-61), antioxidants (pages 61-62), peroxisome proliferators-activated receptors (PPAR) agonists (pages 63-67), cholesterol-lowering agents (pages 68-75); amyloid inhibitors (pages 75-77), amyloid formation inhibitors (pages 77-78), metal chelators (pages 78-79), anti-psychotics and anti-depressants (pages 80-82), nutritional supplements (pages 83-89) and compounds increasing the availability of biologically active substances in the brain (see pages 89-93) and prodrugs (pages 93 and 94), which document is incorporated herein by reference, but especially the compounds mentioned on the pages indicated above.

Such combination therapies noted above encompass combined administration (where two or more therapeutic agents are included in the same or separate formulations), and separate administration, in which case, administration of the antibody of the invention can occur prior to, simultaneously, and/or following, administration of the additional therapeutic agent or agents. In some embodiments, administration of the anti-Tau antibody and administration of an additional therapeutic agent occur within about one month, or within about one, two or three weeks, or within about one, two, three, four, five, or six days, of each other.

An antibody of the invention (and any additional therapeutic agent) can be administered by any suitable means, including parenteral, intrapulmonary, and intranasal, and, if desired for local treatment, intralesional administration. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. Dosing can be by any suitable route, e.g. by injections, such as intravenous or subcutaneous injections, depending in part on whether the administration is brief or chronic. Various dosing schedules including but not limited to single or multiple administrations over various time-points, bolus administration, and pulse infusion are contemplated herein.

Antibodies of the invention would be formulated, dosed, and administered in a fashion consistent with good medical practice. Factors for consideration in this context include the particular disorder being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of delivery of the agent, the method of administration, the scheduling of administration, and other factors known to medical practitioners. The antibody need not be, but is optionally formulated with one or more agents currently used to prevent or treat the disorder in question. The effective amount of such other agents depends on the amount of antibody present in the formulation, the type of disorder or treatment, and other factors discussed above. These are generally used in the same dosages and with administration routes as described herein, or about from 1 to 99% of the dosages described herein, or in any dosage and by any route that is empirically/clinically determined to be appropriate.

For the prevention or treatment of disease, the appropriate dosage of an antibody of the invention (when used alone or in combination with one or more other additional therapeutic agents) will depend on the type of disease to be treated, the type of antibody, the severity and course of the disease, whether the antibody is administered for preventive or therapeutic purposes, previous therapy, the patient's clinical history and response to the antibody, and the discretion of the attending physician. The antibody is suitably administered to the patient at one time or over a series of treatments. Depending on the type and severity of the disease, about 1 μg/kg to 15 mg/kg (e.g. 0.1 mg/kg-10 mg/kg) of antibody can be an initial candidate dosage for administration to the patient, whether, for example, by one or more separate administrations, or by continuous infusion. One typical daily dosage might range from about 1 μg/kg to 100 mg/kg or more, depending on the factors mentioned above. For repeated administrations over several days or longer, depending on the condition, the treatment would generally be sustained until a desired suppression of disease symptoms occurs. One exemplary dosage of the antibody would be in the range from about 0.05 mg/kg to about 10 mg/kg. Thus, one or more doses of about 0.5 mg/kg, 2.0 mg/kg, 4.0 mg/kg or 10 mg/kg (or any combination thereof) may be administered to the patient. Such doses may be administered intermittently, e.g. every week or every three weeks (e.g. such that the patient receives from about two to about twenty, or e.g. about six doses of the antibody). An initial higher loading dose, followed by one or more lower doses may be administered. However, other dosage regimens may be useful. The progress of this therapy is easily monitored by conventional techniques and assays.

It is understood that any of the above formulations or therapeutic methods may be carried out using an immunoconjugate of the invention in place of or in addition to an anti-Tau antibody.

H. Articles of Manufacture

In another aspect of the invention, an article of manufacture containing materials useful for the treatment, prevention and/or diagnosis of the disorders described above is provided. The article of manufacture comprises a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, IV solution bags, etc. The containers may be formed from a variety of materials such as glass or plastic. The container holds a composition which is by itself or combined with another composition effective for treating, preventing and/or diagnosing the condition and may have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). At least one active agent in the composition is an antibody of the invention. The label or package insert indicates that the composition is used for treating the condition of choice. Moreover, the article of manufacture may comprise (a) a first container with a composition contained therein, wherein the composition comprises an antibody of the invention; and (b) a second container with a composition contained therein, wherein the composition comprises a further cytotoxic or otherwise therapeutic agent. The article of manufacture in this embodiment of the invention may further comprise a package insert indicating that the compositions can be used to treat a particular condition. Alternatively, or additionally, the article of manufacture may further comprise a second (or third) container comprising a pharmaceutically-acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution and dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.

It is understood that any of the above articles of manufacture may include an immunoconjugate of the invention in place of or in addition to an anti-Tau antibody.

III. Examples

The following are examples of methods and compositions of the invention. It is understood that various other embodiments may be practiced, given the general description provided above.

Example 1: Generation of Tau for Immunization Generation of Monomeric Recombinant Tau

The recombinant human Tau construct, 2N4R isoform (amino acids 2-441), was fused to a N-terminal His-tag to facilitate purification and characterization. See, e.g., FIG. 15 . The fusion construct was cloned into the pET52b vector (Novagen) and expressed in E. coli. Cells were harvested and lysed under denaturing condition using 7M guanidinium chloride overnight at 4° C. with stirring. Cell debris was pelleted at 40,000 rpm for 1 hour. The recombinant, His-tagged protein was isolated by nickel affinity chromatography (Ni Sepharose excel affinity resin, GE Healthcare Life Sciences) followed by size-exclusion chromatography (Superdex 200 resin, GE Healthcare Life Sciences) under denaturing condition. Guanidinium chloride was removed by dialyzing the recovered protein into 20 mM IVIES, 50 mM NaCl, and 1 mM TCEP at pH 6.8. The His-tag was subsequently removed using TEV protease, followed by final purification using cation exchange chromatography (Mono S column, GE Healthcare Life Sciences) to remove the cleaved His-tag. The purification buffer contained 0.1% Triton x-114 (v/v) to remove endotoxin. Purified protein was exchanged into PBS with 1 mM TCEP. The purity and monomeric state were analyzed by SDS-PAGE and SEC-MALLS. Identity was confirmed by mass spectrometry. Protein concentration was determined by UV absorption at 280 nm. The final product was free of endotoxin (<0.5 EU/mg), as determined by Kinetic Limulus Amebocyte Lysate (LAL) assay.

Generation of Phosphorylated Tau

Phosphorylated Tau was generated using the Tau 2-441 construct prepared using the method described above. The protein construct was phosphorylated using 0.5 μM PKA kinase (Life Technologies), which phosphorylates serine 409, among other residues. The reaction mixture was incubated with 1 mM ATP, 5 mM MgCl₂, at room temperature for 72 hours. Phosphorylation was confirmed by mass spectrometry. Size-exclusion chromatography (Superdex 75, GE Healthcare Life Sciences) was used to remove the kinase. The purity, monomeric state, and endotoxin level of the phosphorylated protein preparation were analyzed substantially as described above.

In Vitro Oligomerization of Monomeric Tau

Oligomeric Tau was generated using the monomeric Tau 2-441 construct. The monomeric protein was first exchanged into 20 mM N,N-Bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES), 25 mM NaCl, pH 7.4, followed by oligomerization using 75 μM arachidonic acid (Cayman Chemicals) and 18 kDa Heparin (Sigma Aldrich), at equimolar concentration with protein at 37° C. for 3 days. Oligomerization was confirmed by thioflavin T fluorescence assay, dynamic light scattering (DLS), and analytical size-exclusion chromatography. Oligomeric Tau is in some instances referred to as “oligoTau.”

Example 2: Generation of Anti-Tau Antibodies Methods Generation of Hybridomas

Female C57BL/6JOlaHsd (C57BL/6) and BALB/c OlaHsd (Balb/c) wild-type mice (Harlan, USA) were received at 9 weeks of age. Tau knock-out mice (B6.129-Mapttm1Hnd/J; The Jackson Laboratory, USA) were received at 6 and 9 weeks of age. Vaccinations started at 12 to 15 weeks of age. Mice were vaccinated with oligomerized human Tau. Before vaccination, the oligoTau was mixed with one of two adjuvants used in this study, Ribi Adjuvant System (Ribi; Sigma-Aldrich, Switzerland) at 50% v/v, or a combination of CpG single-stranded synthetic DNA oligodeoxynucleotides (CpG; Microsynth, Switzerland) and aluminium hydroxide (Al; Brenntag, Switzerland). Ribi is 2% squalene oil-in-water emulsion containing monophosphoryl lipid A (isolated from Salmonella minnesota) and synthetic trehalose dicorynomycolate (isolated from the cord factor of the Tubercle bacillus) in squalene oil, 0.2% Tween-80, and water.

Mice were vaccinated by subcutaneous injection (s.c.), except groups D and G, which received a combination of intraperitoneal (i.p.) and hock administrations. Mice in group D were administered 50 μg of oligoTau i.p. and 10 μg of oligoTau as hock injection. Mice in group G were administered 8 μg of oligoTau i.p. and 2 μg of oligoTau as hock injection. See Table 2.

For vaccinations containing CpG and Al (CpG/Al) as adjuvant, each injection of 200 μL contained 60 μg (30 nmol) CpG, 1 mg Al, and 50 μg oligoTau. For all study groups, mice were injected on days 0, 14, 35, and 56. Mice used for myeloma fusion (Nanotools, Germany) were additionally vaccinated with three daily booster injections of oligoTau (50 μg per i.p. injection) without adjuvant added.

TABLE 2 Mice and vaccination protocols Total oligoTau dose Vaccination Study group Mouse strain (μg/injection) Adjuvant route A C57BL/6 50 CpG/Al s.c. B C57BL/6 50 Ribi s.c. C Balb/c 50 CpG/Al s.c. D Balb/c 60 CpG/Al hock + i.p. E Balb/c 5 CpG/Al s.c. F Balb/c 50 Ribi s.c. G Tau knock-out 10 Ribi hock + i.p.

Mice were bled and sacrificed one day following the last of three booster injections, and splenocytes were fused with myeloma cells to generate antibody producing hybridomas.

Selection of Hybridomas for Subcloning

For fusions, mice were divided into three groups, for a total of 10 fusions (2 fusions in one group, four fusions in the second group, and four fusions in the third group), generating 299 hybridomas. Viable hybridomas were grown using serum-containing selection media, and the best hybridomas were then selected for subcloning, using ELISA assays for full-length human Tau and oligoTau binding as described below. Following limiting dilution, the final hybridomas were then grown in serum-free medium and media was collected from stable colonies for antibody screening and selection.

ELISA Screening Assays

Serum-free supernatants were harvested from stable hybridomas. The supernatants containing antibodies of interest were then screened by ELISA assays to characterize antibody properties and select antibodies for further development. The ELISA assays were used to determine the following: binding to full-length human Tau (flTau; SignalChem, Canada), binding to hyperphosphorylated flTau (Genentech, USA), binding to oligomeric versus monomeric preparations of flTau, and binding to certain antibody Tau epitope(s). Briefly, 96-well MaxiSorp ELISA plates (Nunc, Denmark) were coated with one of the targets as shown in Table 3.

TABLE 3 Targets used for the ELISA screening assays. Assay ELISA setup Target Binding to Direct Full-length human Tau (flTau) coated at flTau ELISA 1 μg/mL Binding to Direct Full-length human Tau phosphorylated in pTau ELISA vitro using 4 kinases (GSK3β, Cdk5, PKA, and CK1δ; hyperphosphorylated Tau or pTau) purified and coated at 1 μg/mL Epitope Direct Biotinylated 15-mer peptides spanning the 441 mapping ELISA amino acids (aa) of human Tau with 9 aa offset and 6 aa overlap coated at 10 μg/mL on a streptavidin 96-well plate Binding to Capture AVI-tag biotinylated oligomeric and oligoTau ELISA monomeric flTau captured in solution by anti-IgG immobilized antibodies being tested

Coating was done overnight in phosphate-buffered saline (PBS) at 4° C. Plates were washed thoroughly with 0.05% Tween-20/PBS and then blocked with 1% bovine serum albumin (BSA) in 0.05% Tween-20/PBS for 1 hr at 37° C. The antibody contained in the hybridoma supernatant was then added at the indicated dilutions, and incubated for 2 hrs at 37° C. after which the plates were washed as described previously.

For the direct ELISAs, an AP-conjugated anti-mouse IgG secondary antibody (Jackson ImmunoResearch Laboratories, United Kingdom) was added at 1/6000 dilution in 0.05% Tween-20/PBS for 2 hr at 37° C. After the final wash, plates were incubated with p-nitrophenyl phosphate disodium hexahydrate (pNPP; Sigma-Aldrich, Switzerland) phosphatase substrate solution, and read at 405 nm using an ELISA plate reader (Tecan, Switzerland). Results are expressed as optical densities (O.D.).

For the oligoTau and monoTau capture ELISAs, antibodies contained in serum-free sterile hybridoma supernatants were immobilized on an anti-IgG coated plate at 500-fold dilution, followed by the incubation of oligoTau or monoTau, both with site-specific biotinylation via an AVI-tag. The target incubations started at 5 μg/mL and then were diluted 8- or 16-fold. Streptavidin-HRP and ABTS substrate was used for signal quantitation in a plate reader (Tecan, Switzerland). Results are expressed as O.D.

Affinity Estimates

Affinity of non-purified antibodies in serum-free hybridoma supernatants was estimated by surface plasmon resonance using a Biacore T-100 instrument (GE Healthcare, United Kingdom). Antibodies were immobilized onto an anti-IgG biosensor chip, and flTau (SignalChem, Canada) was used as the target analyte. Kinetic analysis was done using a 1:1 Langmuir fit model.

SDS-PAGE and Western-Blot Assays

The binding of selected panTau antibodies to Tau in human brain was tested in a Western-blot (WB) using brain lysates from three AD and two age-matched non-AD control donors (Tissue Solutions, United Kingdom). The lysates were processed to obtain a detergent-free soluble Tau fraction. Processed lysates were loaded onto 4-12% bis-tris gels (Novex, Life Technologies, Switzerland) and transferred onto Immobilon PVDF membranes and blotted with antibodies being tested with and an IRDye 800CW goat anti-mouse secondary antibody (Li-Cor, USA).

ELISA Assay Using Human Brain Lysates

To assess the binding of selected antibodies to non-denatured human Tau in AD and control brain lysates, antibodies from hybridoma supernatants, or a negative and positive control antibodies, were immobilized on a 96-well plate as described above. Tau in soluble human brain lysates from AD or age-matched control subjects (400 μg/mL protein; all from Tissue Solutions, United Kingdom) was then captured and detection was performed using a polyclonal rabbit panTau antibody (AbCam, United Kingdom) followed by an Fc-γ fragment specific anti-rabbit IgG-AP (Jackson ImmunoResearch, USA). Brain lysate from Tau knock-out mouse was used as a negative sample control. Plates were incubated with pNPP (Sigma-Aldrich) phosphatase substrate solution, and read at 405 nm using an ELISA plate reader (Tecan, Switzerland). Results are expressed as optical densities (O.D.).

Sequencing of Antibody Hybridomas

Hybridoma cell lysates were supplied to Antitope (Antitope, United Kingdom) for variable region gene sequencing. Briefly, RT-PCR was performed using degenerate primer pools for murine signal sequences together with constant region primers for each of IgG variable heavy (VH), IgM VH, Ig kappa variable light (KVL) and Ig VL chains. Heavy chain V-region mRNA was amplified using a set of six degenerate primer pools (HA to HF) specific for VH signal sequences together with either IgM or IgG-specific constant region primers. The light chain V-region mRNA was amplified using a set of eight signal sequence-specific degenerate primer pools, seven for the κ cluster (KA to KG) and one for the λ cluster (LA), together with either κ or λ constant region primers. The PCR products obtained from successful amplification were purified, cloned into a ‘TA’ cloning vector (pGEM-T Easy, Promega), transformed into E. coli and individual colonies sequenced. The nucleotide and amino acid sequences of the antibody VH and VL regions were determined with the sequences for 27 antibody hybridomas.

Results Selection of Hybridomas for Subcloning

Hybridomas that were generated from each of the three rounds of fusions, a total of 299 hybridomas derived from ten fusions, were initially assayed for binding to flTau, with selected hybridomas additionally assayed for binding to pTau and oligomerized Tau. The aim was to select antibodies that bind equally well to Tau and to Tau modified post-translationally, such as phosphorylated or oligomeric Tau. For this, assays were run on hybridomas to select for the best panTau properties. To determine antibody binding region and the specific Tau epitope, the binding region was first determined using different Tau fragments and then a library of 15-mer overlapping Tau peptides spanning the full 441 amino acids (aa) sequence of the longest human Tau isoform. A group of antibodies binding to pre-determined regions of Tau were intentionally avoided with the aim to maximize binding to different post-translationally modified forms of Tau and to all the six different human Tau isoforms present in humans.

The three fusion series resulted in the generation of 133 subcloned stable hybridomas that were screened for the best panTau properties. A combination of different screening assays was used to narrow down the number of antibody hybridomas having the preferred properties for a panTau antibody. For comparing flTau and pTau binding, 90 hybridomas were assayed with the results of 24 hybridomas shown in FIG. 1A-F. As an initial screen had been performed using Tau fragments to avoid selecting antibodies binding to regions of Tau known to have high density of residues that are phosphorylated in Alzheimer's disease (AD) and other tauopathies, most antibodies tested bound to both flTau and pTau with similar binding properties as determined by this ELISA.

In some embodiments, it is desirable that a panTau antibody bind to both monomeric and oligomeric forms of Tau without a strong preference to one or the other. A capture ELISA was set up to determine if antibodies bound to both monomeric and oligomeric forms of flTau. An ELISA run in capture mode preserves the oligomer conformation of pre-oligomerized Tau and the monomeric state of monoTau better than when run as a direct ELISA with the targets immobilized onto an ELISA plate.

Each assay was run by directly comparing the binding of the two forms of Tau to all 90 antibodies tested. Antibodies known to have preferred binding to either oligoTau or that do not discriminate between the two forms of Tau were used as controls in each assay. The results of 18 hybridomas are shown in FIG. 2A-E.

Mapping the epitopes is important for selecting antibodies with good panTau properties, as antibodies that bind to regions with high density of potential pTau residues (Ser, Thr, and Tyr) can be avoided. Binding to all six isoforms of human Tau was also used as a selection criterion for a panTau antibody. The panTau epitopes of antibodies that had been initially selected were verified and determined with improved accuracy using a library of 49 peptides each having 15 amino acids (aa) spanning the full length of human Tau, with an overlap of 6 aa residues and an offset of 9 aa. The residue numbers are based on the longest isoform of human Tau (441 aa). Non-purified antibodies were used at high 1/10 dilution to verify binding versus no binding to all peptides. Screening of antibodies from 112 hybridomas previously selected by ELISA indicated binding to 20 different Tau epitopes (Table 4).

TABLE 4 Tau epitopes for antibodies Antibody Tau epitope (aa) 14F5-D9  1-15 94B2-B12  1-15 94B2-C1  1-15 10A1-A6 10-24 10A1-D8 10-24 11E10-B8 10-24 17G12-C11 10-24 17G12-D5 10-24 19H6-A1 10-24 19H6-F7 10-24 19H6-G8 10-24 37D3-H12 10-24 37D3-H9 10-24 37E8-B4 10-24 37E8-C2 10-24 3A4-H4 10-24 3H10-E12(A) 10-24 3H10-G12 10-24 44B7-A9 10-24 44B7-B1 10-24 54C1-H11 10-24 61E7-B11 10-24 61H10-B4 10-24 61H10-H3 10-24 127G7-A5 10-24 127G7-E7 10-24 115A4-A3 10-24 115A4-B1 10-24 125B11-B6 10-24 73C8-A5 10-24 73C8-G4 10-24 76B4-D9 10-24 76B4-H7 10-24 123E9-B3 19-33 15C6-A7 19-33 19F8-B1 19-33 24A11-D5 19-33 63H3-B2 19-33 63H3-D8 19-33 64B9-E11 19-33 64B9-F12 19-33 45D2-C9 19-33 45D2-F4 19-33 72E12-B2 19-33 72G10-A7 19-33 72G10-B6 19-33 123E9-A1 19-42 19F8-C11 19-42 7A11-C12 19-42 89F4-A2 28-42 89F4-A1 28-44 12A10-E8 37-51 55E7-B12 37-51 72E12-H9 37-51 55E7-F11 37-51 30D12-B5 64-78 21C1-D8 64-78 21C1-G6 64-78 30D12-F6 64-78 31A3-A4 64-78 31A3-A7 64-78 77D1-D2 64-78 77D1-E6 64-78 30A1-C9 73-87 30A1-D11 73-87 28F5-G8 82-96 28F5-H8 82-96 33G9-A11 100-114 33G9-B9 100-114 52F2-E12 100-114 52F2-E8 100-114 52F6-B3 100-114 52F6-F11 100-114 56D3-C8 100-114 56D3-E9 100-114 70B10-B6B2 100-114 70B10-B6G12 100-114 78E4-D11 100-114 78E4-G4 100-114 30G1-B2 109-123 30G1-C11 109-123 49G10-F4 109-123 49G10-H1 109-123 65B1-A2 109-123 65B1-A7 109-123 73H6-B8 109-123 113F5-A8 109-123 113F5-F7 109-123 125B11-H3 109-123 26C1-B11 118-132 26C1-C8 118-132 74H10-A3 118-132 74H10-C3 118-132 78F3-B2 118-132 78F3-E7C6 118-132 78F3-E7H7 118-132 126H12-G6 136-150 126H12-H7 136-150 22G7-C9 154-168 22G7-G9 154-168 111B8-C4 163-177 111B8-F10 163-177 66F5-A1 172-177 66F5-F2 172-177 71H8-A1 190-204 71H8-D6 190-204 83E10-D10 190-204 83E10-D6 190-204 126F11-B3 217-231 126F11-G11 217-231 93A8-C9 397-411 93A8-D2 397-411

For affinity measurements to flTau, 46 antibodies were measured using SPR on a Biacore instrument, with the K_(DS) determined. Biacore affinity measurements were done by immobilizing antibodies on an anti-IgG chip and using flTau as the target analyte. Results for 32 antibodies are shown in Table 5, with antibodies ranked based on affinity to flTau. Of the antibodies measured for affinity to flTau, 22 antibodies had affinities better than 20 nM, of which 14 antibodies had K_(DS) under 5 nM with antibody 37D3-H9 having a K_(D) (affinity) of 1 nM.

TABLE 5 Affinity for flTau Antibody K_(D) (nM) 37D3-H9 1 54C1-H11 1.5 123E9-A1 1.8 94B2-C1 1.9 24A11-D5 2 113F5-F7 2.4 89F4-A1 2.9 19F8-B1 2.9 61E7-C4 3.3 126F11-G11 4.2 26C1-C8 4.3 93A8-D2 4.3 37E8-B4 4.4 61E7-B11 4.8 125B11-H3 6 54C1-C3 6.8 3A4-H4 7.8 52F6-F11 8.4 3A4-A12 10.1 44B7-B1 14.7 3H10-E12 19.4 10A1-D8 19.6 52F2-E8 26 19H6-F7 39 34H4-F5 43 19H6-A1 56 34H4-B10 69 17G12-C11 118 45H12-C4 139 17G12-D5 161 61H10-H3 177 11E10-C3 399

To verify the binding of selected antibodies to all six isoforms of human Tau, an SDS-PAGE was run with a recombinant Tau ladder containing all six isoforms and Western-blot (WB) done using three selected Tau antibodies. All three panTau antibodies bind to all six Tau isoforms (FIG. 3 ). Furthermore, brain homogenates from three AD and two age-matched controls were simultaneously run for comparison. As expected, and based on the mapped epitopes, all three antibodies tested in this assay showed binding to all six Tau isoforms. The difference observed in band patterns between human AD and control donors may represent the greater phosphorylation and/or SDS-stable Tau aggregates that would be expected to be present in AD subjects.

Human Alzheimer's disease (AD) and control samples were additionally run in a non-denaturing ELISA capture assay to verify binding to Tau in human brains. Samples lysates processed for soluble Tau from two AD and two non-AD age-matched control subjects were run at 8 dilutions testing three antibodies (FIG. 4A-C).

Antibody variable chain sequences were determined for 27 hybridomas (Antitope, United Kingdom). Protein sequences for certain heavy and light chain variable domains and hypervariable regions (HVRs) are shown in the Table of Sequences.

Example 3: Characterization Anti-Tau Antibodies

Antibody heavy and light chains were constructed via gene synthesis and subcloning of the resulting DNA into murine IgG2a (heavy chain) and murine kappa (light chain) mammalian expression vectors. Antibodies were expressed in CHO or 293T cells by transient co-transfection of the heavy chain and light chain plasmids and were purified with affinity resin MabSelectSure (GE Healthcare Life Sciences). Purified recombinant antibodies were screened for binding to Tau monomer protein on a Biacore T200 surface plasmon resonance instrument using a mouse IgG capture kit and a Series S CM5 chip. Antibodies in mIgG2a format diluted in 10 mM HEPES pH7.4, 150 mM NaCl, 0.05% Tween 20 (running buffer, HBSP) were captured for 30 or 45 seconds at a concentration of 1 μg/ml (antibodies 26C1, 94B2-C1, 52F6-F11.v1, 52F6-F11.v2, 11E10-B8, 55E7-F11, 125B11-H3, 123E9-A1, 30G1-B2, 66F5-A1, 89F4-A1, 93A8-D2 and 126F11-G11) or for 70 or 150 seconds at a concentration of 0.1 μg/ml (antibodies 19H6-F7, 3A4-H4, 54C1-H11 and 37D3-H9) using a flow rate of 10 μl/min. Binding of Tau monomer in HBSP was monitored at 25° C. using a flow rate of 30 μl/min and concentrations of 16, 31, 63, 125, 125, 250 and 500 nM for antibodies 26C1 and 94B2; 16, 31, 63, 125, 125, 250, 500 and 1000 nM for antibodies 52F6-F11.v1 and 52F6-F11.v2; 6, 19, 56, 56, 167 and 500 nM for antibodies 11E10-B8, 55E7-F11 and 125B11-H3; 5, 16, 49, 148, 148, 444, 1333 and 4000 nM for antibodies 123E9-A1, 30G1-B2, 66F5-A1, 89F4-A1, 93A8-D2 and 126F11-G11; 0.4, 1.6, 6.3, 2.5, 100 and 400 nM for 19H6-F7; and 0.2, 0.8, 4, 4, 20 and 100 nM for 3A4-H4, 54C1-H11 and 37D3-H9. Association and dissociation times were monitored for 180-480 seconds and for 300-600 seconds respectively. Antibody 37D3-H9 was selected for further analysis due to the high affinity (Table 6) and the absence of NXS/T glycosylation motifs in the CDRs.

TABLE 6 K_(D) (nM) of murine antibodies to human Tau monomer. Data shown represent output of a 1:1 binding model. Antibody K_(D) (nM) k_(on) (1/Ms) k_(off) (1/s) 26C1 17 4 × 10⁴ 7 × 10⁻⁴ 94B2-C1 6 5 × 10⁴ 3 × 10⁻⁴ 54C1-H11 0.6 3 × 10⁵ 2 × 10⁻⁴ 3A4-H4 12 3 × 10⁴ 3 × 10⁻⁴ 37D3-H9 1.6 1 × 10⁵ 1 × 10⁻⁴ 19H6-F7 10 2 × 10⁵ 2 × 10⁻³ 11E10-B8 108 2 × 10⁵ 2 × 10⁻² 55E7-F11 171 2 × 10⁵ 4 × 10⁻² 125B11-H3 5 5 × 10⁴ 3 × 10⁻⁴ 123E9-A1 52 4 × 10⁵ 2 × 10⁻² 30G1-B2 20 4 × 10⁵ 8 × 10⁻³ 66F5-A1 105 8 × 10⁴ 8 × 10⁻³ 89F4-A1 27 3 × 10⁵ 7 × 10⁻³ 93A8-D2 6 3 × 10⁵ 2 × 10⁻³ 126F11-G11 3 2 × 10⁶ 4 × 10⁻³ 52F6-F11.v1 15 5 × 10⁴ 7 × 10⁻⁴ 52F6-F11.v2 5 7 × 10⁴ 4 × 10⁻⁴ 37D3-H9 Demonstrates Avidity when Binding to Tau Protein

Human monomer Tau protein was covalently coupled to a Biacore Series S CM5 chip using the Biacore Amine Coupling Kit (GE Life Sciences), resulting in immobilization to a level of approximately 128 RU. Direct binding of 37D3-H9 in both Fab and IgG formats was monitored using the single-cycle kinetics experimental format with five association periods of 300 s each and antibody concentrations of 1, 2, 4, 8 and 16 nM (IgG) or 5, 10, 20, 40 and 80 nM (Fab). Dissociation was monitored for 7200 seconds (Fab) or for 14400 seconds (IgG). A value for the dissociation rate was calculated by fitting a 1:1 binding model to the data. Calculated dissociation rates were 5.0×10⁻⁴ for 37D3-H9 Fab and 1.1×10⁻⁵ for 37D3-H9 IgG, a 45-fold difference. FIG. 5 illustrates the difference in the dissociation rates of Fab (left panel) and IgG (right panel), indicating that 37D3-H9 IgG is demonstrating avidity.

Example 4: Humanization of Anti-Tau Antibodies

Antibody 37D3-H9 was humanized by grafting the antibody CDRs and selected variable region framework residues onto human antibody consensus frameworks (Dennis, M. S. (2010). CDR repair: A novel approach to antibody humanization. In Current Trends in Monoclonal Antibody Development and Manufacturing, S. J. Shire, W. Gombotz, K. Bechtold-Peters and J. Andya, eds. (Springer, New York), pp. 9-28). Grafting onto consensus VH3, Vκ2 and Vκ1 frameworks was assessed. The heavy chain graft included murine residue at position 49 (Kabat numbering system). The Vκ2 graft included murine residues in framework positions 2 and 4. The Vκ1 graft included murine residues in framework positions 2, 4 and 43. Humanized variants were constructed by gene synthesis and subcloning into human IgG₁ or IgG₄ and Kappa chain mammalian expression vectors. Antibodies were expressed by co-transfection of the heavy and light chain plasmids into CHO cells and purified with affinity resin MabSelect Sure. Humanized variants were screened for affinity to human Tau monomer using the Biacore human IgG capture kit, a Series S CM5 chip and a Biacore T200 instrument. Antibodies were diluted to 2 μg/ml and captured for 15 seconds at 10 μl/min. Association and dissociation of 100, 33, 11 and 3.7 nM human Tau monomer in 10 mM HEPES pH7.4, 150 mM NaCl, 0.05% Tween 20 (running buffer, HBSP) was monitored for 180 seconds and 600 seconds respectively at a flow rate of 30 μl/min. A 1:1 binding model was applied to the results (Table 7).

TABLE 7 Affinity screening of humanized variants for monomeric human Tau Antibody Light chain variant framework K_(D) (nM) hu37D3-H9.v1 Kappa1 4.1 hu37D3-H9.v2 Kappa1 5.6 hu37D3-H9.v3 Kappa1 8.8 hu37D3-H9.v4 Kappa1 8.2 hu37D3-H9.v5 Kappa2 1.9 hu37D3-H9.v6 Kappa2 3.5 hu37D3-H9.v7 Kappa2 27.0 hu37D3-H9.v8 Kappa2 10.2 hu37D3-H9.v9 Kappa2 13.2 hu37D3-H9.v10 Kappa2 14.3 hu37D3-H9.v11 Kappa2 74.8 hu37D3-H9.v12 Kappa2 21.6 hu37D3-H9.v13 Kappa2 9.0 hu37D3-H9.v14 Kappa2 10.8 hu37D3-H9.v15 Kappa2 19.0 hu37D3-H9.v16 Kappa2 27.2 hu37D3-H9.v17 Kappa2 8.1 hu37D3-H9.v18 Kappa2 13.4 hu37D3-H9.v19 Kappa2 55.7 hu37D3-H9.v20 Kappa2 36.9 hu37D3-H9.v21 Kappa2 38.1 hu37D3-H9.v22 Kappa2 36.6 hu37D3-H9.v23 Kappa2 81.1 hu37D3-H9.v24 Kappa2 56.6

Antibody variants hu37D3-H9.v1, hu37D3-H9.v2, hu37D3-H9.v5 and hu37D3-H9.v6 were characterized further by surface plasmon resonance with additional antibody concentrations and longer association/dissociation times. These variants were analyzed with a broader range of human Tau monomer concentrations (1.2, 3.7, 11.1, 11.1, 33.3, 100 nM) and increased association (300 seconds) and dissociation (1200 seconds) periods. A 1:1 binding model was applied to the results (Table 8).

TABLE 8 Detailed analysis of binding kinetics of selected variants to human Tau by surface plasmon resonance Antibody Light chain variant framework K_(D) (nM) hu37D3-H9.v1 Kappa1 1.1 nM, 1.0 nM hu37D3-H9.v2 Kappa1 1.2 nM hu37D3-H9.v5 Kappa2 0.8 nM hu37D3-H9.v6 Kappa2 1.4 nM

A YTE (M252Y/S254T/T256E) mutation was incorporated into certain IgG₄ antibodies. Fc Receptor-neonate (FcRn) binding domain mutations such as M252Y, S254T and T256E (YTE) have been described to increase FcRn binding and thus increase the half-life of antibodies. See U.S. Published Patent Application No. 2003/0190311 and Dall'Acqua et al., J. Biol. Chem. 281:23514-23524 (2006).

Antibody 125B11-H3 was humanized onto VH3 and W1 consensus frameworks. The heavy chain graft included murine residues at position 78 (Kabat numbering system). The W1 graft included murine residues in framework positions 43 and 87. The light chain of 113F5-F7 was also humanized onto the W1 framework, with additional murine residues at framework positions 43 and 87. Humanized variant heavy chains (125B11) and light chains (125B11 and 113F5-F7) were co-transfected in multiple combinations and purified in 96-well format as described above. Humanized variants were then screened for affinity for human Tau monomer using the Biacore human IgG capture kit, a Series S CM5 chip and a Biacore T200 instrument. Antibodies were diluted to 2 μg/ml and captured for 15 seconds at 10 l/min. Association and dissociation of 0, 100 and 500 nM human Tau monomer in HBSP was monitored for 180 s and 300 s respectively at a flow rate of 40 l/min. A 1:1 binding model was applied to the results (Table 9).

TABLE 9 Screening of 125B11-H3 and 113F5-F7 humanization variants by surface plasmon resonance 125B11 heavy chain humanization variant Screening K_(D) (nM) HC1 HC2 HC3 HC4 HC5 HC6 125B11 light LC1 16, 19 18 18 15 85 — chain LC2 20 20 19 14 —* NT humanization LC3 21 23 20 15 — — variant LC4 23 22 20 17 >100 >100 113F5-F7 LC1 57 61 54 44 — — light chain LC2 67 68 55 47 — — humanization LC3 61 64 54 47 >100 — variant LC4 71 77 65 51 — — *Minimal binding to Tau monomer. NT, not tested.

Variants hu125B11.v17 (HC3+LC1), hu125B11.v26 (HC4+LC2) and hu125B11.v28 (HC4+LC4) were selected for high-resolution kinetic analysis based on the affinity screen (Table 10). Antibody 94B2-C1 was humanized onto VH1 and Vκ2 frameworks. The heavy chain graft also included murine residues at position 28, 29, 67, 69, 71, and 73 (Kabat numbering system). The Vκ2 graft also included murine residues in framework positions 2, 36, and 46. Combinations of eight heavy chains and eight light chains were expressed, purified and screened by surface plasmon resonance (SPR) as described for 125B11 above. Results of the SPR screen are shown in Table 11. Variant hu94B2.v105 (heavy chain variant 94B2.HCl, light chain variant 94B2.LC13) was selected for detailed SPR characterization (Table 11).

TABLE 10 Kinetic data for selected humanized anti-Tau antibody variants Antibody Isotype K_(D) (nM) k_(on) (1/Ms) k_(off) (1/s) hu125B11.v17 hIgG1 10.5 0.8 × 10⁵ 0.8 × 10⁻³ hu125B11.v26 hIgG1 9.5 0.7 × 10⁵ 0.7 × 10⁻³ hu125B11.v28 hIgG1 10.2 0.7 × 10⁵ 0.7 × 10⁻³ hu94B2.v105 hIgG1 3.7 0.8 × 10⁵ 0.3 × 10⁻³

TABLE 11 Screening of 94B2 humanization variants by surface plasmon resonance 94B2 Light Chain humanization variant: Screening K_(D) (nM) LC9 LC10 LC11 LC12 LC13 LC14 LC15 LC16 94B2 Heavy HC1  3.8* ^(§) 91.5 ^(§)  4.1^(¶) ^(§) 104.0 ^(§) Chain HC2 5.7 ^(§) 89.6 ^(§) 7.4 NT 99.6 ^(§) humanization HC3 2.0 ^(§) 69.3 ^(§) 3.8 ^(§) 64.1 ^(§) variant: HC4 61.9  ^(§) ^(§) ^(§) 64.1  ^(§) ^(§) ^(§) HC5 2.7 ^(§) 62.6 ^(§) 4.0 ^(§) 72.6 ^(§) HC6 0.9 ^(§) 70.1 ^(§) 3.0 ^(§) 74.1 ^(§) HC7 52.9  ^(§) ^(§) ^(§) 57.8  ^(§) ^(§) ^(§) HC8 1.0 ^(§) 44.3 ^(§) 2.4 ^(§) 51.5 ^(§) *Mean of n = 3 repeats. ^(¶)hu94B2.v105. ^(§)Minimal binding to Tau monomer observed. NT, not tested.

Example 5: Stability Analysis of Humanized Anti-Tau Antibodies Identification of Chemical Instability

Antibody samples were thermally stressed to mimic stability over the shelf life of the product. Samples were buffer exchanged into 20 mM Acetate buffer, pH 5.5, or phosphate buffer, pH 7.4, and diluted to a concentration of 1 mg/ml. One ml of sample was stressed at 40° C. for 2 weeks and a second was stored at −70° C. as a control. Both samples were then digested using trypsin to create peptides that could be analyzed using liquid chromatography (LC)-mass spectrometry (MS) analysis. For each peptide in the sample retention time, from the LC as well as high resolution accurate mass and peptide ion fragmentation information (amino acid sequence information) were acquired in the MS. Extracted ion chromatograms (XIC) were taken for peptides of interest (native and modified peptide ions) from the data sets at a window of ±10 ppm and peaks were integrated to determine area. Relative percentages of modification were calculated for each sample by taking the (area of the modified peptide) divided by (area of the modified peptide plus the area of the native peptide) multiplied by 100. These relative percentages were then compared between the control (t=0) and the stressed (t=2 weeks) samples. Percentages shown represent the control (t=0) value subtracted from the stressed (t=2 weeks) value. Deamidation analysis of antibodies hu37D3-H9.v1 and hu37D3-H9.v5 led to the observation that the sequence N²⁸G²⁹N³⁰ (Kabat numbering) within the light chain CDR-1 was susceptible to deamidation. The increase in deamidated N²⁸G²⁹N³⁰ was found to be 16.5% for hu37D3-H9.v1 and 11% for hu37D3-H9.v5.

Impact of Deamidation on Antibody Binding to Antigen

To assess the impact of N²⁸ deamidation on the affinity for human Tau, it was desirable to obtain two samples with widely separated N²⁸ deamidation status. Hu37D3-H9.v5 hIgG4.S228P was incubated at 40° C. for two weeks at a concentration of 1 mg/ml in Phosphate Buffered Saline, pH 7.4. Deamidation of the N²⁸G²⁹ motif was measured using LC-MS/MS. The t=2 week stressed sample had a 43.1% increase deamidation relative to the t=0 unstressed sample. The stressed and unstressed antibodies were analyzed for Tau binding by surface plasmon resonance (Biacore) using the GE Biacore human IgG capture kit and a Series S CM5 chip. The hIgG were diluted to 2 μg/ml in 10 mM HEPES pH7.4, 150 mM NaCl, 0.05% Tween 20 (running buffer, HBSP) and captured at a flow rate of 10 μl/min for 15 seconds (t0 sample) or 17 seconds (t2 sample). Kinetic data was collected for Human Tau monomer injected at concentrations of 0, 3.1, 6.3, 12.5, 25, 25, 50 & 100 nM in HBSP, using a flow rate of 30 μl/min, a 300 s association phase and an 1800 s dissociation phase. Between cycles the surface was regenerated using a 30 second injection of 3M Magnesium Chloride at 10 μl/min. A 1:1 binding model was fitted to the data using instrument defaults, including local fitting of the “RI” parameter. Results shown in FIG. 6 and Table 12 demonstrate that although the stressed antibody immobilized at greater levels than the unstressed antibody in this experiment, the magnitude of the Tau binding signal (as represented by the magnitude of the parameter Rmax) was noticeably lower. After normalizing the Rmax value for the differences in capture level, the stressed (t=2 weeks) sample appeared to show approximately half the total Tau binding capacity of the unstressed sample (indicated by a 56% reduction in the Normalized Rmax). The calculated affinity did not appear to change: in this analysis the difference in K_(D) between the t=0 and the t=2 weeks samples was less than 2% (K_(D)=0.7 nM for t=0 and t=2 weeks). The results are consistent with the t=2 weeks sample containing a significantly reduced population of high affinity antibody.

TABLE 12 Relative binding of stressed and unstressed hu37D3-H9.v5 samples to monomeric Tau by surface plasmon resonance hu37D3-H9.v5 Ligand Normalized Rmax Change in hIgG4.S228P Level Rmax (=Rmax/Ligand Normalized sample (RU) (RU) Level) Rmax Control (t = 0) 102.9 47.7 0.46 N/A Stressed (t = 2 weeks) 146.8 30.2 0.21 −56%

Impact of Deamidation on Antibody Binding to Antigen and Calculation of “Normalized Rmax”

Given that asparagine deamidation is expected to result in aspartic acid and iso-aspartic acid products (Bischoff R. & Kolbe H. V. J. (1994). J. Chromat. 5, 662, p 261-278) the impact of replacing N²⁸ with D²⁸ (variant hu37D3-H9.v5 N28D) on affinity for human Tau monomer was analyzed. Affinity was assessed at 25° C. using a Biacore T200 instrument, the GE Biacore human IgG capture kit and a CM5 Series S chip. The hIgG were diluted to 2 μg/ml in 10 mM HEPES pH7.4, 150 mM NaCl, 0.05% Tween 20 (running buffer, HBSP) and captured at a flow rate of 10 μl/min for 22 seconds. Kinetic data was collected for human Tau monomer injected at concentrations of 0, 6.3, 12.5, 25, 25, 50, 100, 200, and 400 nM in HBSP, using a flow rate of 30 μl/min, a 300 second association phase and a 600 second dissociation phase. Between cycles the surface was regenerated using a 30 second injection of 3M Magnesium Chloride at 10 μl/min. A 1:1 binding model was fitted to the data and affinities for hu37D3-H9.v5 and hu37D3-H9.v5.3 (also referred to herein as hu37D3-H9.v5 N28D) calculated using kinetic analysis. Parameters used for the 1:1 fitting included the Instrument default of local fitting for the “RI” parameter. The results are shown in FIG. 7 and Table 13.

Calculated K_(D) for the hu37D3-H9.v5 N28D variant was 160×10⁻⁹ M, compared to 1.5×10⁻⁹ M (mean, n=4 intra-experiment determinations) for hu37D3-H9.v5 analyzed under the same conditions. Therefore, conversion of N²⁸ to D²⁸ causes >100-fold reduction in affinity. Given the comparatively low affinity of the hu37D3-H9.v5 N28D variant, and the comparatively rapid kinetics, we reasoned that the kinetics analysis of a mixture of the N²⁸ and D²⁸ variants would be dominated by the higher affinity population, and that presence of the lower affinity variants might be reflected by a reduction in the Normalized Rmax. To validate this reasoning, the Tau-binding profile of antibody variants hu37D3-H9.v5 and hu37D3-H9.v5 N28D were compared to that of the two antibodies mixed together in equal quantities. Compared to hu37D3-H9.v5 alone, a 1:1 mix of hu37D3-H9.v5 and hu37D3-H9.v5 N28D resulted in a 45% reduction in Normalized Rmax (Table 13). We concluded that changes in Normalized Rmax upon thermal stress may be indicative of a reduced population of high affinity antibody in the stressed sample. We reasoned that changes in Normalized Rmax could therefore be used to screen variants of hu37D3-H9 for improved stability.

TABLE 13 Changes in Normalized Rmax observed upon thermal stress of hu37D3-H9.v5 and upon mixing of hu37D3-H9.v5 with anticipated deamidation product hu37D3-H9.v5 N28D Decrease in Normalized Rmax K_(D) Rmax compared to Sample (nM) (RU) Reference* Comments hu37D3-H9.v5 1.5 ± 0.2 76.1 ± 0.4 Reference Mean +/− Standard hIgG1 Deviation of four intra- experiment analyses hu37D3-H9.v5 N28D 160 81.0  4% hIgG1 hu37D3-H9.v5 & 2.0 46.4 45% Two antibodies hu37D3-H9.v5 N28D mixed at a 1:1 ratio hIgG1 hu37D3-H9.v5 1.5 68.8  3% Control for hIgG4.S228P, Stressed sample t = 0 hu37D3-H9.v5 1.5 33.4 54% Stressed sample hIgG4.S228P, t = 2 weeks *Normalized Rmax = Rmax (RU)/Ligand Level (RU). Normalized Rmax for reference antibody = 0.33 (mean of four intra-experiment determinations, standard deviation <0.01).

Antibody Optimization and Selection

Ninety 37D3-H9 variants were assessed by Biacore to compare their functional stability with or without a two-week 40° C. thermal stress period. The variants included most single mutations of the N²⁸G²⁹N³⁰T³¹ motif, double mutants containing the G29A mutation, double mutations of Asn-28 and Tyr-32 that might functionally replace these to hydrogen-bonded residues, as well as all possible permutations of residues 2, 4, 33, and 93 as either the residues present in the original 37D3-H9 antibody or the corresponding germline residue variant. In addition, mutations were tested in the context of residue 1 being Asp or Glu, which does not impact affinity or stability of the Asn-28 residue.

Antibodies were expressed by transient transfection of Expi293 cells in 96-well format and automated purification performed on a Tecan freedom EVO 200 liquid handling system with a 500 μL MCA96 head. Briefly, IgGs in 1 mL culuture were captured using tip columns that were custom packed with 20 μL Mab Select SuRe resin (Glygen Corp & GE Healthcare). After washing with 1×PBS pH 7.4, IgGs were eluted into 160 μL of 50 mM phosphoric acid pH 3 and neutralized with 12 μL of 20×PBS pH 11. Mab Select SuRe tip columns were stripped in 0.1 M NaOH and regenerated with 1×PBS pH 7.4 for consecutive use of up to 15 times. Purified antibodies in 96-well format were normalized to 0.1 mg/ml using a Hamilton Star liquid handling robot. The “pre-stress” samples were kept at approximately 4° C. and the “post-stress” samples were incubated at 40° C. for two weeks in a PCR machine. Functional stability of the variants was compared by running surface plasmon resonance kinetics experiments with the “pre-stress” and “post-stress” antibody preps. The antibodies were assessed using a human antibody capture CM5 Series S chip generated using the GE Biacore human IgG capture kit and a Biacore T200 instrument. Antibodies diluted to 2 μg/ml were immobilized using a 15 second injection time and 10 μl/min flow rate. Binding to Tau monomer at 0 nM, 26.5 nM and 265 nM, at 25° C., using a flow rate of 40 μl/min, was monitored for a 180 second association phase followed by a 300 second dissociation phase. Samples were run in 10 mM HEPES pH7.4, 150 mM NaCl, 0.05% Tween 20 (HBSP) using a multi-cycle kinetics format. Data was analyzed using BIAevaluation software, fitting a 1:1 binding model. The resulting affinity (K_(D)) values are shown in FIG. 8A-D. A Stability Index was also calculated, using the rationale that affinity-compromised antibodies (due for example to deamidation of key residues) are expected to contribute equally to the IgG capture level (“Ligand Level”) but to contribute less to the measured Tau binding, and that this would be reflected in the experimentally derived value for Rmax. To account for variations in the amount of each antibody captured, Rmax was normalized for the antibody capture level (as measured by “Ligand Level”, Response Units immobilized during antibody capture). Thus Normalized Rmax is calculated as the experimental Rmax (units=RU) divided by the “Ligand Level” (Evaluation output representing the RU captured during the hIgG capture step, units=RU), and Stability Index is calculated here as Normalized Rmax (post-stress) divided by Normalized Rmax (pre-stress).

Selected antibodies were expressed by transient transfection of CHO cells and purified. The antibodies were then stressed for two weeks at 1 mg/ml and deamidation analyzed by LC-MS/MS, using RCM tryptic peptide mapping with DTT reduction, IAA capping and pH 8.2 digestion. Results (Table 14) demonstrated that variant hu37D3-H9.v28.A4 had reduced susceptibility to deamidation on the N²⁸G²⁹N³⁰ motif. The reduced deamidation of the hu37D3-H9.v28.A4 was unexpected, as the residue is not located in the immediate vicinity of the Asn-28 residue (FIG. 9 ) and it is not clear how the F33L mutation might stabilize Asn-28.

TABLE 14 Stability of the hu37D3-H9.v28.A4 variants in stress tests for deamidation Increase in deamidation Thermal Stress of light chain Antibody Conditions N²⁸G²⁹N³⁰ hu37D3-H9.v1 hIgG1 40° C. in Acetate 16.5% Buffer, pH 5.5 hu37D3-H9.v5 hIgG1 40° C. in Acetate   11% Buffer, pH 5.5 hu37D3- hIgG1 40° C. in Acetate N²⁸: 2.8% H9.v28.A4 Buffer, pH 5.5 N³⁰: 0.2% 37° C. in PBS N²⁸: 5.3% pH 7.4 N³⁰: ND hIgG4.S228P.YTE 40° C. in Acetate N²⁸: 0% Buffer, pH 5.5 N³⁰: 0% 37° C. in PBS N²⁸: 10.4% pH 7.4 N³⁰: 2.0%

Example 6: Humanized Anti-Tau Antibody Selection and Characterization Antibody Selection and Characterization: Binding to Human Tau Protein

Affinity of selected antibodies was assessed at 25° C. using a Biacore T200 instrument, the GE Biacore human IgG capture kit and a CM5 Series S chip. The hIgG were diluted to 0.25 μg/ml in 10 mM HEPES pH7.4, 150 mM NaCl, 0.05% Tween 20 (running buffer, HBSP) and captured at a flow rate of 10 l/min for 150 seconds. Kinetic data was collected for Human Tau monomer injected at concentrations of 0, 0.4, 1.2, 3.7, 11, 11, 33 and 100 nM in HBSP, using a flow rate of 30 l/min, a 300 second association phase and a 600 second dissociation phase. Between cycles the surface was regenerated using two sequential 30 second injections of 3M MgCl at 10 l/min. Data was fit to a 1:1 binding model (Table 15).

TABLE 15 Kinetic data for selected humanized anti-Tau antibody variants K_(D) k_(on) k_(off) Antibody Isotype (nM) (1/Ms) (1/s) hu37D3- hIgG1 1.5 6.9 × 10⁵ 1.1 × 10⁻³ H9.v28.A4 hu37D3-H9.v5 hIgG1 1.0 7.5 × 10⁵ 0.8 × 10⁻³ hu37D3-H9.v5 hIgG4.S228P 1.3 7.1 × 10⁵ 0.9 × 10⁻³ hu37D3-H9.v1 hIgG4.S228P 2.0 6.7 × 10⁵ 1.3 × 10⁻³ Antibody Characterization: Binding to Human Tau Protein in hIgG4.S228P.YTE Format

Affinity was assessed at 25° C. using a Biacore T200 instrument, the GE Biacore human FAb capture kit and a CM5 Series S chip. The hIgG were diluted to 0.5 μg/ml in 10 mM HEPES pH7.4, 150 mM NaCl, 0.05% Tween 20 (running buffer, HBSP) and captured at a flow rate of 10 μl/min for 180 seconds. Kinetic data was collected for Human Tau monomer injected at concentrations of 0, 0.4, 1.2, 3.7, 11, 11, 33 and 100 nM in HBSP, using a flow rate of 30 μl/min, a 300 second association phase and a 600 second dissociation phase. Between cycles the surface was regenerated using two sequential 60 second injections of 10 mM Glycine pH 2.1. Data was fit to a 1:1 binding model. Kinetic data are shown in Table 16.

TABLE 16 Binding kinetics of hu37D3-H9.v28.A4 hIgG4.S228P.YTE to monomeric human Tau by surface plasmon resonance Antibody K_(D) k_(on) k_(off) Antibody preparation (nM) (1/Ms) (1/s) hu37D3-H9.v28.A4 Prep 1 1.4 6 × 10⁵ 9 × 10⁻⁴ hIgG4.S228P.YTE Prep 2 1.4 6 × 10⁵ 9 × 10⁻⁴

Antibody Characterization: Binding to Cynomolgus Monkey Tau Protein

Affinity was assessed at 25° C. using a Biacore T200 instrument, the GE Biacore human IgG capture kit and a CM5 Series S chip. The hIgG were diluted to 2 μg/ml in 10 mM HEPES pH7.4, 150 mM NaCl, 0.05% Tween 20 (running buffer, HBSP) and captured at a flow rate of 10 μl/min for 15 seconds. Kinetic data was collected for Human Tau monomer injected at a minimum of five different non-zero concentrations between 1.2 and 100 nM, with one replicate concentration. Kinetics were assessed using a flow rate of 30 μl/min, a 300 second association phase and a 600 second dissociation phase. Between cycles a 30 second regeneration injection of 3M Magnesium Chloride was performed at a flow rate of 10 μl/min. The results were fit to a 1:1 binding model. Kinetic data are shown in Table 17.

TABLE 17 Affinity of humanized anti-Tau antibodies for monomeric cynomolgus monkey Tau Ligand Level Rmax K_(D) k_(on) k_(off) Antibody (RU) (RU) (nM) (1/Ms) (1/s) hu37D3.v28.A4 113.9 62.6 0.7 17 × 10⁵ 1 × 10⁻³ hu37D3.v28.F1 126.9 61.2 1.3 12 × 10⁵ 2 × 10⁻³ hu37D3.v28.A12 162.6 85.2 1.0 17 × 10⁵ 2 × 10⁻³ hu37D3.v29.2 168.6 86.0 1.4 17 × 10⁵ 2 × 10⁻³ hu37D3-H9.v5 125.1 55.5 0.6 15 × 10⁵ 1 × 10⁻³ hu37D3-H9.v1 130.2 51.7 0.8 20 × 10⁵ 1 × 10⁻³

Humanized antibodies hu37D3.v28.A4 and hu37D3.v28.F1 also bind to phosphorylated Tau (pTau).

Example 7: Pharmacokinetics of Anti-Tau Antibody

To evaluate the pharmacokinetics of the anti-Tau 37D3-H9 mIgG2a antibody in vivo, C57BL/6 mice were administered a single intravenous (IV) or intraperitoneal (IP) bolus injection at a dose of 10 mg/kg to conscious mice. At various time points up to 28 days post-dose, plasma samples were collected to determined anti-Tau antibody concentrations.

The concentrations of the dosed antibody in mouse plasma were measured with a generic ELISA using a mouse anti-muIgG2a antibody coat, followed by adding plasma samples starting at a dilution of 1:100, and finished by adding a mouse anti-muIgG2a-biotin conjugate, and then streptavidin conjugated to horseradish peroxidase for detection. The assay had a standard curve range of 1.56-200 ng/mL and a limit of detection of 0.16 μg/mL. Results below this limit of detection were reported as less than reportable (LTR).

FIG. 10 shows the results of the pharmacokinetic analysis for anti-Tau 37D3-H9 mIgG2a. Anti-Tau 37D3-H9 mIgG2a had similar exposure and clearance in wild-type C57BL/6 mice as isotype control antibodies, with a clearance of 6.31 mL/day/kg.

To evaluate the pharmacokinetics of anti-Tau 94B2-C1 mIgG2a and anti-tau 125B11-H3 mIgG2a in vivo, a single IP bolus injection of antibody was administered at a dose of 10 mg/kg to conscious C57BL/6 mice. At various time points up to 28 days post-dose, plasma samples were collected to determined anti-Tau antibody concentrations.

The concentrations of the dosed antibody in mouse plasma and was measured with a generic ELISA using a mouse anti-muIgG2a antibody coat, followed by adding plasma samples starting at a dilution of 1:100, and finished by adding a mouse anti-muIgG2a-biotin conjugate, and then streptavidin conjugated to horseradish peroxidase for detection. The assay had a standard curve range of 0.78-100 ng/mL and a limit of detection of 0.078 μg/mL. The concentrations were also measured with a specific ELISA using recombinant Tau as the coat, followed by adding plasma samples starting at a dilution of 1:10, and finished by adding goat anti-mIgG2a conjugated to horseradish peroxidase for detection. The assay had a standard curve range of 0.078-10 ng/mL and a limit of detection of 0.0008 μg/mL. Results below this limit of detection were reported as less than reportable (LTR).

The results of those experiments are shown in FIGS. 16 and 17 . Anti-Tau 94B2 mIgG2a had similar exposure and clearance in wild-type C57BL/6 mice as an isotype control antibody when concentrations were analyzed using a generic assay, but lower exposure and faster clearance when concentrations were analyzed using a specific assay. See FIG. 16 . The clearance determined by the generic assay was 4.06 mL/day/kg and that determined by the specific assay was 7.53 mL/day/kg. These results suggest that the antibody may undergo in vivo changes over time that compromise its ability to recognize its target. Anti-Tau 125B11-H3 mIgG2a had similar exposure and clearance in wild-type C57BL/6 mice as an isotype control antibody, regardless of which assay generated the concentrations. See FIG. 17 . The clearance determined by the generic assay is 4.96 mL/day/kg and that determined by the specific assay is 4.90 mL/day/kg.

Table 18 shows the pharmacokinetic parameters for anti-Tau antibodies 37D3-H9, 94B2-C1, and 125B11-H3 in mice.

TABLE 18 Pharmacokinetic parameters for anti-Tau antibodies Admin- istration Cmax AUCinf CL or CL/F Route Assay (μg/mL) (μg/mL*day) (mL/day/kg) 37D3- IV Generic 185 1590 6.31 H9 IP Generic 107 1680 6.76 94B2- IP Generic 151 2460 4.06 C1 IP Specific 141 1330 4.91 125B11- IP Generic 127 2020 4.96 H3 IP Specific 151 2040 4.90

To evaluate the pharmacokinetics of hu37D3.v28.A4 hIgG4.S228P and hu37D3.v28.A4 hIgG4-S228P.YTE antibodies in vivo, cynomolgus monkeys (Macaca fascicularis) were administered a single IV bolus injection at a dose of 1 mg/kg to conscious moneys. At various time points up to 49 days post-dose, plasma samples were collected to determined anti-Tau antibody concentrations.

The concentrations of the dosed antibody in monkey plasma and was measured with a generic ELISA using a sheep anti-human IgG antibody coat, followed by adding plasma samples starting at a dilution of 1:100, and finished by adding goat anti-human IgG conjugated to horseradish peroxidase for detection. The assay had a standard curve range of 0.156-20 ng/mL and a limit of detection of 0.02 μg/mL. Results below this limit of detection were reported as less than reportable (LTR).

FIG. 11 shows the results of the pharmacokinetic analysis for hu37D3.v28.A4 hIgG4.S228P and hu37D3.v28.A4 hIgG4-S228P.YTE. In FIG. 11 , each set of datapoints represents one animal and the lines represent the average for all animals in the antibody and assay group. Table 19 shows the pharmacokinetic parameters for hu37D3.v28.A4 hIgG4.S228P and hu37D3.v28.A4 hIgG4-S228P.YTE in cynomolgus monkeys.

TABLE 19 Pharmacokinetic parameters for hu37D3.v28.A4 hIgG4.S228P and hu37D3.v28.A4 hIgG4-S228P.YTE in cynomolgus monkeys Cmax AUCinf CL Vss Antibody Assay (μg/mL) (day*μg/mL) (mL/day/kg) (mL/kg) anti-gD hlgG4 Generic 34.6 386 2.66 55.5 hu37D3.v28.A4 Generic 35.7 ± 2.59 559 ± 209  1.97 ± 0.743 71.9 ± 16.0 hIgG4.S228P Specific 35.4 ± 1.37 419 ± 89.9 2.47 ± 0.581 60.8 ± 3.49 hu37D3.v28.A4 Generic 34.5 ± 5.23 578 ± 43.5 1.74 ± 0.125 60.5 ± 1.87 hIgG4.S228P.YTE Specific 33.5 ± 2.72 520 ± 39.0 1.93 ± 0.139 56.5 ± 4.90

Example 8: Further Epitope Characterization of Anti-Tau Antibody

Following a comparison of 37D3-H9 binding to biotinylated Tau monomer and biotinylated peptide (MAPT_10-24), binding of 37D3-H9 to additional biotinylated peptides was also assessed. Nunc maxisorp 96-well microplates were coated at 4° C. for >12 hours with Neutravidin diluted to 2 μg/ml in 50 mM Sodium Carbonate Buffer, pH 9.6. All subsequent incubations were performed at room temperature. After coating, plates were blocked with Superblock™ (PBS) Blocking Buffer (Thermo Fisher Scientific) for two hours then washed thoroughly with PBS, 0.05% Polysorbate 20. Wells were then exposed to biotinylated Tau peptides (Table 20) or Avi-tag biotinylated Tau monomer at 1 μg/ml for one hour and washed as previously. Peptides were synthesized using standard solid-phase Fmoc chemistry (see, e.g., Fmoc solid phase peptide synthesis: A practical approach; Chan, W. C., White, P. D., Eds.; Oxford University Press: New York, 2000). Antibodies 37D3-H9 mIgG2a and hu37D3-H9.v5 hIgG1, serially diluted from 500 nM to 50 pM in 90% Superblock™ (PBS) Blocking Buffer, were allowed to bind biotinylated-Tau coated wells for 90 minutes. Wells were washed as previously and bound antibody detected with peroxidase-conjugated secondary antibody (Invitrogen/Life Technologies) diluted 1/1000 in Superblock™ Blocking Buffer (Rabbit anti-Mouse IgG or Goat Anti-Human IgG (H+L) respectively). After twenty minutes wells were washed as previously and signal developed with TMB Microwell 2-Component Substrate (KPL). Reactions were stopped by addition of 1M Phosphoric Acid and absorbance at 450 nm was measured with a SpectraMax M2 platereader.

TABLE 20 Peptide Sequences MAPT SEQ ID sequence Peptide sequence NO: MAPT(10-24) VMEDHAGTYGLGDRK 592 MAPT(2-24) AEPRQEFEVMEDHAGTYGLGDRK 593 MAPT(2-34) AEPRQEFEVMEDHAGTYGLGDRKDQGGYT 594 MHQD MAPT(10-44) VMEDHAGTYGLGDRKDQGGYTMHQDQEGD 595 TDAGLK

The results of that experiment are shown in FIG. 12 . FIG. 12A shows binding of each of the indicated antibodies for the indicated peptides. Antibodies 37D3-H9 and 94B2-C1 both showed strong binding to fragment 10-24 in that experiment, and antibody 94B2-C1 also showed strong binding to fragment 1-15. Antibodies 19F8-C11 and 123E9-A1 showed strong binding to fragment 19-33, while antibody 89F4-A1 showed strong binding to fragments 28-42 and 37-51. See FIG. 12A. Antibodies 37D3-H9 mIgG2a and hu37D3-H9.v5 hIgG1 both showed strong binding to Tau fragments 2-24 and 2-34 and weaker binding to fragment 10-24. See FIGS. 12B and 12C. These results suggest that antibodies 37D3-H9 mIgG2a and hu37D3-H9.v5 hIgG1 bind an epitope of Tau within amino acids 2-24 of the mature protein.

In an alanine scanning substitution experiment, mutations Y18A and L20A were found to abrogate binding by murine antibody 37D3-H9 to a Tau fragment (fragment 2-21), suggesting that the antibody contacts these Tau residues. Using a series of 15mer offset peptides, it was found that murine antibody 37D3-H9 showed similar binding to fragment 9-23 as to fragment 10-24, and also showed moderate binding to fragments 7-21, 8-22, and 11-25.

Example 9: Cell-Based Characterization of 37D3-119 Humanized Antibodies Methods Primary Hippocampal and Microglial Culture and Hippocampal-Microglial Co-Culture

Dissociated primary hippocampal neurons were prepared from embryonic day 16-17 wild-type C57BL/6N mice. Cells were plated onto PDL/laminin-coated 8-well chamber slides (Biocoat, 354688 Corning) at 25,000 cells/well. Cells were plated and maintained in NbActiv4 (BrainBits) and half of the media was replaced twice a week. Recombinant tau and antibodies were applied to the culture at 18 cell divisions.

For microglial culture, cortices and hippocampi from postnatal day 1-2 C57BL/6N mice were dissociated and grown in 10% FBS in DMEM in 225 mm² culture flasks for 10-12 days. The culture flasks were gently shaken to dissociate microglia and the cells in 10% FBS in DMEM were replated onto either PDL/laminin-coated 8-well chamber slides at 30,000 cells/well for imaging or uncoated 48-well plates (3548, Corning) at 100,000 cells/well for cytokine assay. 4-5 hours after plating, cells were switched to serum-free low-glucose DMEM and maintained overnight before treatment with recombinant tau and antibodies.

Hippocampal-microglial co-cultures were prepared by replating microglia dissociated from 225 mm² culture flasks onto 18 DIV primary hippocampal neurons in 8-well slide chambers (12,500 microglia and 25,000 neurons per one well). Co-cultures were treated with recombinant tau and antibodies 4 hours after microglia plating.

In Vitro Treatment of Recombinant Tau and Antibodies

For 18 DIV hippocampal cultures or hippocampal-microglial co-cultures, recombinant human oligomeric tau and antibodies (500 nM each at 1:1 ratio) or controls were pre-incubated in neuron culture medium (conditioned medium from 18 DIV hippocampal culture:fresh NbActiv4 at 1:1) for 1 hour at 37° C. before they were added to the cells. Cells were incubated with the tau-antibody mix or control in the media for 72 hours (hippocampal culture) or 48 hours (hippocampal-microglial co-culture). Cells were washed with PBS three times before fixation.

For microglia culture, recombinant human oligomeric tau and antibodies or controls were pre-incubated at 125 nM each (immunocytochemistry/imaging) or 250 nM each (cytokine assay) in low-glucose DMEM in the absence of serum for 1 hour at 37° C. prior to the addition to the cells. For immunocytochemistry/imaging, cells were incubated with the tau-antibody mix or controls for 10 minutes and washed three times with PBS before fixation. For cytokine assay, cells were incubated with the tau-antibody mix or control for 24 hours and medium of each well was collected for cytokine assay.

Immunocytochemistry, Imaging, and Quanitificaton

Cells were fixed with 4% paraformaldehyde in PBS for 15 min and permeabilized with 0.1% Triton X-100 in PBS for 10 minutes. 10% donkey serum was used for blocking and cells were incubated with primary antibodies in PBS overnight at 4° C., followed by incubation with Alexa-fluorophore-labeled secondary antibodies against appropriate species developed in donkey (Invitrogen). Primary antibodies used were anti-tau (DAKO), rabbit anti-human tau developed against the human tau N-terminal region spanning amino acids 11-24, anti-MAP2 (ab5392, Abcam), and anti-Iba-1 (ab5076, Abcam). The slides were mounted with Prolong Gold DAPI (P36935, Invitrogen) and no. 1 coverslips.

Confocal fluorescent imaging was performed with a LSM780 (Carl Zeiss, Inc.) using Zen 2010 software (Carl Zeiss, Inc.). For imaging of hippocampal cultures and hippocampal-microglial co-cultures, 5 z-stack images at 0.98 μm intervals were collected using Plan Apochromat 20×/0.8 M27 objective lens. For the MAP2 fragmentation assay, a maximum intensity z projection was created for the image stack and analyzed using Metamorph (Molecular Devices, Sunnyvale, Calif.). A median filter and nearest neighbor deconvolution were used for noise reduction. Neurite and cell body lengths were analyzed using the neurite outgrowth module followed by morphological processing. Fragments less than 15 pixels (6.225 μm) were normalized to total signal length to obtain a measure of MAP2 fragmentation.

Microglia were imaged with α-Plan Apochromat 100×/1.46 M27 objective. Quantification of recombinant tau uptake in the cells was performed with Image J (1.43u, 64-bit, National Institute of Health). ROIs of cell area were drawn manually using Iba-1 signal as a reference. Area and integrated intensity of tau immunoreactivity of ROI were measured to obtain tau immunoreactivity normalized to area. All analyses were performed blinded to experimental conditions.

Results

The results of the experiment are shown in FIG. 13 . As shown in FIG. 13A, antibodies with full effector function were not protective against Tau toxicity in the neuron-microglia co-cultures. FIG. 13B shows images of neuron-microglia co-cultures contacted with oligomeric Tau and antibodies (bottom panels). Antibody 37D3-H9 hIgG4 and hu37D3-H9 hIgG1 (N297G), which lack effector function, were protective against Tau toxicity, while 37D3-H9 hIgG1 was not.

Example 10: Dose-Dependent Reduction of Tau Pathology in Tau Tg Mice Administered 37D3-H9 IgG₂a or 37D3-H9 IgG2a DANG

Transgenic mice expressing human Tau P301L under the Thy1 promoter (Tau P301L-Tg) were maintained on a C57BL/6N (Charles River) background. Tau P301L-Tg and wild type littermate mice were assigned to treatment groups and dosed once weekly intraperitoneally (i.p.) with either IgG2a-control (anti-gp120) at 30 mg/kg, anti-tau 37D3-H9 WT IgG2a at 3, 10 or 30 mg/kg, anti-tau 37D3-H9 DANG IgG2a at 3, 10 or 30 mg/kg. DANG refers to D265A/N297G mutations in IgG2a, which abrogate effector function. All antibody-dosing solutions were prepared in 10 mM histidine pH 5.8, 6% sucrose, 0.02% Tween 20 at a concentration of 10 mg/ml. Treatment started at 13 weeks of age. The mouse groups in the in vivo study were males and staggered into 3 cohorts. In addition, 3 TauP301L-Tg mice were harvested at age 3 months without undergoing any treatment in order to determine the baseline level of pathology at the time of treatment initiation.

To harvest tissue, mice were anesthetized with 2.5% tribromoethanol (0.5 ml per 25 g body weight) and transcardially perfused with PBS. Brains were harvested and bisected. Right hemispheres were fixed in 4% paraformaldehyde overnight at 4° C. then transferred to phosphate buffered saline prior to processing for immunohistochemistry. Left hemispheres were sub-dissected on ice then frozen at −80° C. for biochemical analysis. Tail clips were taken from all mice to confirm genotypes.

Hemibrains were multiply embedded into a gelatin matrix using MultiBrain® blocks (Neuroscience Associates, Knoxville, Tenn.) and sectioned coronally at 25 μm thickness. Within each block, the brain position was randomized relative to genotype and treatment. Free-floating sections of individual mouse hemibrains or of MultiBrain® blocks were stained as previously described (Le Pichon et al., 2013, PLoS One, 8(4): e62342), but with washes in PBS instead of Iris buffered saline and primary antibody incubations at 4° C. instead of room temperature. Primary antibody was rabbit anti-pTau212/214 (generated in-house; 0.01 μg/ml). To avoid high background staining, in the case of mouse primary antibodies that were subtype specific, we used the corresponding subtype-specific secondary antibody (eg. Biotinylated anti-mouse IgG3, Bethyl A90-111B).

Immunohistochemically stained slides were imaged using the Leica SCN400 (Leica Microsystems; Buffalo Grove, Ill.) whole slide scanning system at 200× magnification with a resolution of 0.5 μm/pixel, Regions of interest (ROIs) were manually drawn on 4 matched hippocampal levels per animal, and the amount of staining in these ROIs was quantified in an automated fashion using the two endpoints described below. All image analysis was performed blind to genotype and treatment groups. For positive pixel area analysis for quantitation of RIC stains, digital images of antibody-labeled brain sections were analyzed as previously described (Le Pichon et al., 2013). The percent area stained was calculated by normalizing the total positive pixels to the total pixel area of the ROI. The integrated intensity was calculated using the Beer-Lambert law, absorbance=−log(transmitted light intensity/incident light intensity), for the positive pixel areas only.

The results of that experiment are shown in FIG. 14 . Administration of anti-tau 37D3-H9 WT IgG2a or anti-tau 37D3-H9 DANG IgG2a resulted in a dose-dependent reduction of pTau212/214 in the hippocampus.

Example 11: Humanized 37D3-H9 Kappa 1 Variants

Humanized antibody variants based on hu37D3-H9.v1, which has a kappa 1 light chain, were made and tested for N²⁸ stability. An alignment of the light chain variable region of the three variants tested with hu37D3-H9.v1 is shown in FIG. 18 . The three variants differ from each other in the light chain variable region: hu37D3.v39 contains the mutation F33L, hu37D3.v40 contains the mutation G29T and hu37D3.v41 contains the mutation N30Q.

Antibody samples were thermally stressed, as follows. Samples were buffer exchanged into 20 mM histidine acetate, 240 mM sucrose, pH 5.5 and diluted to a concentration of 1 mg/ml. One ml of sample was stressed at 40° C. for 2 weeks and a second was stored at −70° C. as a control. Both samples were then digested using trypsin to create peptides that could be analyzed using liquid chromatography (LC)-mass spectrometry (MS) analysis. For each peptide in the sample retention time, from the LC as well as high resolution accurate mass and peptide ion fragmentation information (amino acid sequence information) were acquired in the MS. Extracted ion chromatograms (XIC) were taken for peptides of interest (native and modified peptide ions) from the data sets at a window of ±10 ppm and peaks were integrated to determine area. Relative percentages of modification were calculated for each sample by taking the (area of the modified peptide) divided by (area of the modified peptide plus the area of the native peptide) multiplied by 100. These relative percentages were then compared between the control (t=0) and the stressed (t=2 weeks) samples. Percentages shown represent the control (t=0) value subtracted from the stressed (t=2 weeks) value. The results are shown in Table 21. The results demonstrate that the F33L mutation is effective for reducing deamidation in a kappa 1 humanized light chains.

TABLE 21 Stability of the hu37D3-H9.v1 variants in stress tests for deamidation. Increase in deamidation of Antibody light chain N²⁸G²⁹N³⁰ hu37D3.v39 hIgG4.S228P.YTE N²⁸: 2.7% N³⁰: No significant increase detected hu37D3.v40 hIgG4.S228P.YTE N²⁸: 12.1% N³⁰: 3.9% hu37D3.v41 hIgG4.S228P.YTE N²⁸: 6.0% N³⁰: Residue replaced with glutamine

Affinity of the humanized antibody variants was measured at 25° C. using a Biacore T200 instrument, the GE Biacore human FAb capture kit, and a CM5 Series S chip. Antibodies were diluted to 1 μg/ml in HBSP (10 mM HEPES pH7.4, 150 mM NaCl, 0.05% Tween 20) and captured at a flow rate of 10 l/min for 180 seconds. Kinetic data were collected for human Tau monomer injected at 1.2, 3.7, 11, 33 and 100 nM in HBSP using the Single Cycle Kinetics methodology and a flow rate of 30 l/min. Each concentration of Tau monomer was injected for a period of 3 minutes and dissociation was monitored for ten minutes. Between cycles, the surface was regenerated with two sequential one-minute injections of 10 mM glycine pH2.1. Data was fit to a 1:1 binding model using BIAEvaluation software. Each antibody was analyzed twice within the experiment; data in Table 22 are shown as mean±range.

TABLE 22 Affinities of hu37D3-H9.v1 variants for monomeric Tau K_(D) k_(on) K_(off) (nM) (1/Ms) (1/s) hu37D3.v1 hIgG1 2.3 ± 0.3 6 ± 0.5 × 10⁵ 1 ± 0.1 × 10⁻³ hu37D3.v1 hIgG4 2.3 ± 0.3 6 ± 0.2 × 10⁵ 1 ± 0.1 × 10⁻³ hu37D3.v39 hIgG4.YTE 1.9 ± 0.2 6 ± 0.6 × 10⁵ 1 ± 0.02 × 10⁻³  hu37D3.v40 hIgG4.YTE 4.4 ± 0.5 8 ± 0.9 × 10⁵ 3 ± 0.02 × 10⁻³  hu37D3.v41 hIgG4.YTE 5.4 ± 0.3 9 ± 1.2 × 10⁵ 5 ± 0.3 × 10⁻³

Example 12: Pharmacokinetics and Pharmacodynamics of hu37D3.v28.A4 hIgG4-S228P and hu37D3.v28.A4 hIgG4-S228P.YTE in Cynomolgus Monkeys

To evaluate the pharmacokinetics and pharmacodynamics of hu37D3.v28.A4 hIgG4.S228P and hu37D3.v28.A4 hIgG4-S228P.YTE antibodies in vivo, five conscious cynomolgus monkeys (Macaca fascicularis) per group were administered a single IV bolus injection at a dose of 50 mg/kg in the first phase. Anti-gD hIgG4 was used as a control, also at a dose of 50 mg/kg. At various time points up to 35 days post-dose, plasma and CSF samples were collected to determine anti-Tau antibody concentrations. After the final sample collection, the animals were allowed to recover for 63-64 days before initiation of the second phase. In the second phase, the 15 animals from the first phase, plus 3 additional animals, were divided into two groups; the first group (n=9) was administered antibody hu37D3.v28.A4 hIgG4.S228P and the second group (n=9) was administered hu37D3.v28.A4 hIgG4-S228P.YTE antibody, both at 50 mg/kg. Brains of 4 or 5 animals per group were harvested at 2 days and 10 days post-dose.

Human IgG4 antibodies in cynomolgus monkey plasma, CSF, and brain homogenate (described below) were measured with an ELISA using a sheep anti-human IgG monkey adsorbed antibody coat, followed by adding plasma samples starting at a dilution of 1:100, CSF samples starting at a dilution of 1:20, or brain homogenate samples starting at a dilution of 1:10, and finished by adding a goat anti-human IgG antibody conjugated to horseradish peroxidase monkey adsorbed for detection. Color was developed using 3,3′,5,5′-tetramethylbenzidine and neutralized using 1M phosphoric acid. Samples were read at 450/620 nm. The assay has a standard curve range of 0.156-20 ng/mL and a limit of detection of 0.02 ug/mL for plasma, 0.003 μg/mL for CSF, and 0.002 μg/mL for brain homogenate. Results below this concentration were reported as less than reportable (LTR).

The results of the pharmacokinetic analysis are shown in FIG. 19A (plasma) and 19B (CSF), and in Tables 23 and 24. Animals that were suspected of being anti-therapeutic antibody positive (ATA+) were excluded from the analysis. These data show that introducing the YTE mutations in the Fc region of hu37D3.v28.A4 hIgG4.S228P slowed the peripheral and CSF clearance rates of the antibody by about two fold.

TABLE 23 Mean (±SD) plasma clearance and C_(max) estimates following single IV bolus dose Plasma clearance C_(max) Antibody (mL/day/kg) (μg/mL) anti-gD hIgG4 1.67 ± 0.415 1950 ± 174 hu37D3.v28.A4 hIgG4.S228P 2.09 ± 0.229 1970 ± 144 hu37D3.v28.A4 hIgG4-S228P.YTE 1.12 ± 0.233 1850 ± 156

TABLE 24 Mean (±SD) CSF C_(max) estimates following single IV bolus dose C_(max) Antibody (μg/mL) anti-gD hIgG4  1.39 ± 0.751 hu37D3.v28.A4 hIgG4.S228P 0.910 ± 0.552 hu37D3.v28.A4 hIgG4-S228P.YTE 2.51 ± 1.93

The brain concentration of the antibodies at 2 and 10 days post-injection was determined as follows. Brain tissue was weighed and then homogenized in 1% NP-40 in phosphate-buffered saline containing cOmplete™, Mini, EDTA-free protease inhibitor cocktail tablets. The homogenized brain samples were then rotated at 4° C. for 1 hour before spinning at 14,000 rpm for 20 minutes. The supernatant was isolated for brain antibody measurement by ELISA, as described above. The results of that experiment are shown in FIGS. 21A-D. The concentration of antibody hu37D3.v28.A4 hIgG4-S228P.YTE in the brain, and the ratio of brain:plasma concentration for antibody hu37D3.v28.A4 hIgG4-S228P.YTE trended higher than antibody hu37D3.v28.A4 hIgG4. S228P.

The pharmacodynamics response in plasma was also determined. The concentration of total Tau in K₂EDTA plasma was determined using an electrochemiluminescence (ECL) immunoassay (Roche Professional Diagnostics (RPD), Penzberg, Germany). The Elecsys® immunoassay is validated for the quantification of total Tau in human CSF, and because of the similarity between human and cynomolgus monkey Tau, was considered acceptable for the measurement of cynomolgus monkey Tau in CSF and plasma. The assay captures and detects amino acids 159-224 of human and cynomolgus monkey Tau, a region present in all known isoforms, independent of phosphorylation state. The lower detection limit (LDL) of the assay is 1.01 pg/mL. The assay is tolerant to 15.0 mg/mL of hu37D3.v28.A4 hIgG4-S228P.YTE.

The results of the pharmacodynamic analysis are shown in FIG. 20 . There were 3 animals per group after excluding animals suspected of being ATA+, and another animal that lacked baseline values. Surprisingly, within the first day of dosing, plasma Tau levels rise to a greater degree in the animals treated with the YTE variant versus the non-YTE variant. Further, that result is not predicted from the pharmacokinetics response (FIG. 20 ), as the PK is similar between the variants at the early time points. A more robust response is sustained in the animals treated with the YTE variant for the entire duration of sampling.

Example 13: Pharmacokinetics and Pharmacodynamics of hu37D3.v28.A4 hIgG4-S228P.YTE in Cynomolgus Monkey Brain

To assess antibody pharmacokinetics in brain, twelve conscious cynomolgus monkeys (Macaca fascicularis) per group were administered a single IV bolus injection of hu37D3.v28.A4 hIgG4-S228P.YTE at a dose of 50 mg/kg. Anti-gD hIgG4 was used as a control, also at a dose of 50 mg/kg. At various time points up to 42 days post-dose, plasma samples were collected to determine anti-Tau antibody concentrations. In addition, at various time points up to 42 days, 2 monkeys were sacrificed and brain and CSF concentrations of antibody were determined.

Antibody concentrations were determined substantially as described in Example 12.

FIG. 22A-B show the concentration of antibody in cynomolgus monkey brain at various time points post-dose, plotted in logarithmic (A) and linear (B) scale. Table 25 shows the brain concentration parameters.

TABLE 25 Mean (±SD) brain PK parameter estimates following single IV bolus dose Cmax AUCall Group (μg/ml) (day*μg/ml) anti-gD hIgG4 0.175 ± 0.02 4.26 ± 0.35 hu37D3.v28.A4 hIgG4-S228P.YTE  0.12. ± 0.03 3.88 ± 0.89 The hu37D3.v28.A4 hIgG4-S228P.YTE antibody showed increased brain concentration at the terminal timepoint, compared to anti-gD.

The concentration of the antibodies in various regions of the brain, including hippocampus, cerebellum, and frontal cortex, was also determined. FIG. 23A-C and Tables 26 to 28 show the results of that analysis.

TABLE 26 Mean hippocampus PK parameter estimates following single IV bolus dose Cmax AUCall Group (μg/ml) (day*μg/ml) anti-gD hIgG4 0.159 3.95 hu37D3.v28.A4 hIgG4-S228P.YTE 0.087 2.87

TABLE 27 Mean cerebellum PK parameter estimates following single IV bolus dose Cmax AUCall Group (μg/ml) (day*μg/ml) anti-gD hIgG4 0.196 4.30 hu37D3.v28.A4 hIgG4-S228P.YTE 0.139 4.56

TABLE 28 Mean frontal cortex PK parameter estimates following single IV bolus dose Cmax AUCall Group (μg/ml) (day*μg/ml) anti-gD hIgG4 0.17 4.65 hu37D3.v28.A4 hIgG4-S228P.YTE 0.138 4.22

The results of that experiment show exposure of various regions of the brain to antibody hu37D3.v28.A4 hIgG4-S228P.YTE following a single IV injection. Overall exposures in brain were comparable across the two groups, however, similar to the observations in plasma, there was about a two-fold increase in antibody concentrations in the brain at the terminal timepoint in animals dosed with antibody hu37D3.v28.A4 hIgG4-S228P.YTE, compared to anti-gD. See FIG. 23 . These results suggest maintenance of higher trough (terminal) concentrations in brain after dosing with the YTE antibody.

The concentration of the antibodies in CSF and plasma over time was also determined. FIGS. 23D (CSF) and 23E (plasma) and Tables 29 and 30 show the results of that analysis.

TABLE 29 Mean CSF PK parameter estimates following single IV bolus dose Cmax AUCall Group (μg/ml) (day*μg/ml) anti-gD hIgG4 1.270 18.400 hu37D3.v28.A4 hIgG4-S228P.YTE 3.980 21.100

TABLE 30 Mean plasma PK parameter estimates following single IV bolus dose Terminal Cmax Tmax AUCall (Day 43) Group (μg/ml) Day (day*μg/ml) μg/mL anti-gD hIgG4 0.175 ± 0.02 2 4.26 ± 0.35 36.3 ± 14.1 hu37D3.v28.A4  0.12. ± 0.03 3 3.88 ± 0.89 89.4 ± 42.3 hIgG4-S228P.YTE

Again, similar to the plasma and brain pharmacokinetics, there was about a two-fold increased antibody concentration in CSF and plasma at the terminal timepoint in animals dosed with antibody hu37D3.v28.A4 hIgG4-S228P.YTE, compared to anti-gD. See FIG. 23 .

Using the collected plasma samples from the cynomolgus monkeys, the plasma pharmacodynamics of antibody hu37D3.v28.A4 hIgG4-S228P.YTE and control antibody following a single IV 50 mg/kg dose were assessed. Plasma Tau was quantitated using the Elecsys® immunoassay discussed in Example 12.

The results of the pharmacodynamics analysis are shown in FIG. 24A-B. FIG. 24A shows the mean total plasma Tau concentration, normalized to baseline. FIG. 24B shows the total plasma Tau concentration in individual monkeys in the study, normalized to baseline. Similar to the results observed in Example 12, administration of antibody hu37D3.v28.A4 hIgG4-S228P.YTE resulted in significantly increased plasma Tau levels. While not intending to be bound by any particular theory, these data suggest that hu37D3.v28.A4 hIgG4-S228P.YTE binds to Tau in the brain, and consequently Tau is cleared from the brain into the periphery. These results are consistent with target engagement in the brain by hu37D3.v28.A4 hIgG4-S228P.YTE.

Example 14: Affinity Maturation of Antibody hu37D3.v28.A4

Affinity maturation of the 37D3.v28.A4 was done by deep sequencing of a scanning mutagenesis library. Two libraries were designed, one for each antibody chain, in which selected positions in the heavy or light chain variable regions were randomized with an NNK (IUPAC code) codon, which encodes any amino acid or an amber stop codon. The design allows only one amino acid change in the antibody variable regions per clone. The positions were selected from CDRs and framework positions in direct contact or near to CDRs. Light chain Kabat positions 1 to 5, 24 to 27, 29 to 36, 38, 43, 44, 46 to 58, 60, 63 to 71, 87 and 89 to 97 and heavy chain Kabat positions 1, 2, 4, 24 to 39, 43, 45 to 81, 82a, 82b, 83, 85, 86, 91 and 93 to 103 were randomized with NNK codons. The light chain clones that did not randomize position 28 had a Ser at position 28 instead of the wild-type Asn. Two different DNA fragments were used to randomize position 28 of the light chain. One clone had a codon VNK and the other an NYK codon, which allowed for any amino acid except Tyr, Cys, Trp and stop codons. The libraries were created by DNA synthesis (GeneWiz), producing 60 independent linear DNA fragments for the light chain and 75 for the heavy chain, with one position in each fragment randomized with the NNK codon or the VNK/NYK mix. The linear DNA fragments were pooled for each chain and cloned into a monovalent Fab fragment phage display vector (see Lee C V et al., J. Immunol. Methods 284:119-132 (2004)). The light chain library clones had a wild-type heavy chain variable region whereas the heavy chain library clones had a light chain variable region with an N28S mutation. Ligation products were electroporated into Escherichia coli XL-1, superinfected with M13 KO7 helper phage (New England Biolabs) and grown as described (see Koenig P et al., J. Biol. Chem. 290:21773-21786 (2015)).

Three sequential rounds of sorting were performed, using Tau monomer protein adsorbed to an ELISA plate (Round 1) or a solution of an N-terminally biotinylated peptide encompassing residues 2 to 24 (biotin-PEG-AEPRQEFEVMEDHAGTYGLGDRK; SEQ ID NO: 624) of human Tau (Rounds 2 and 3). In the first round, phage (1 OD₂₆₈/ml) were incubated with a Tau-coated ELISA plate for 2 hours at ambient temperature. In the second and third rounds, phage (5 OD₂₆₈/ml) were incubated with 100 nM biotinylated Tau peptide for 2 hours at ambient temperature, then diluted 5-fold and captured on ELISA plates coated with neutravidin (Pierce) for 10 minutes. Stringency was increased in Round 3 by incubating wells containing captured phage with binding buffer (PBS, 0.5% Bovine Serum Albumin) for an additional 20 minutes after the phage capture and washing. The wash step was then repeated. Phage were eluted from the ELISA plates by incubation with 0.1M hydrochloric acid and were propagated by infection of Escherichia coli followed by superinfection with M13KO7 helper phage (New England Biolabs). To facilitate analysis by next-generation sequencing, in Round 3 a “mock” selection was performed in parallel with the selection just described. The “mock” selection, intended to provide a negative control or reference sample, was performed using the same method as the Round 3 just described but omitting the addition of biotinylated peptide.

Initial results were obtained by Sanger sequencing of individual colonies.

In addition, plasmid DNA was extracted from the Escherichia coli XL-1 populations used for phage amplification. Inserts were amplified by an 18-cycle PCR amplification using Phusion DNA polymerase (New England Biolabs) followed by agarose gel purification of amplicons. Amplicons were sequenced by Illumina sequencing as previously described (Koenig P., et al. (2015). 1 Biol. Chem. 290, 21773-21786). Sequences were filtered using the PCR primer sequences and by removing sequences that were longer or shorter than the parental sequence, or contained more than one coding mutation in the variable region. Enrichment ratios were calculated by dividing the frequency of a given mutation at a given position in one population by the frequency of the very same mutation in a second population. Populations were named using the following abbreviations:

-   -   R0—Unsorted library     -   R2—Phage from the Round 2 selection using 100 nM biotinylated         peptide     -   R3—Phage from the Round 3 selection using 100 nM biotinylated         peptide     -   R3M—Phage from the Round 3 “mock” selection.

Selected mutations were transferred, either individually or in combination, onto the hu37D3.v28.A4 variable domains, synthesized and cloned into an IgG mammalian cell expression vector. In some instances, selected mutations were transferred onto variable domains that that had been humanized differently to hu37D3.v28.A4. Examples of alternatively humanized antibodies containing mutations identified by the phage display procedures described above include hu37D3-H9.v76, hu37D3-H9.v83, and hu37D3-H9.v93.

Heavy chain and light chain plasmids were expressed in Expi293 cells by transient transfection and IgG purified from the supernatants using affinity chromatography with resin Mab Select SuRe (GE Life Sciences). Purified IgG were analyzed for binding to human Tau monomer using surface plasmon resonance. Briefly, a Biacore T200 instrument was used to capture antibodies on a human IgG capture chip that had been generated using a Series S CM5 Sensor Chip and the human IgG capture kit (GE Life Sciences). Captured antibodies were exposed to human Tau monomer in solution and the association and dissociation phases monitored. Affinities were calculated by fitting a 1:1 binding model to the kinetic data using Biacore Evaluation software.

A comparison of the light and heavy chain variable regions for hu37D3-H9.v76, hu37D3-H9.v83, and hu37D3-H9.v93 versus the parent hu37D3-H9.v28.A4 is shown in FIGS. 25A-B. Black shading indicates amino acids that are different between the affinity-matured and parent antibody.

The affinity-matured hu37D3-H9.v76, hu37D3-H9.v83, and hu37D3-H9.v93 antibodies were evaluated for their monovalent interaction with human and cynomolgus monkey recombinant Tau monomer.

Affinity for human and cynomolgus monkey Tau was measured at 37° C. using a Biacore T200 instrument, the GE Biacore human IgG capture kit and a Series S CM5 Sensor Chip. Antibodies were diluted to 1 μg/ml in running buffer HBS-EP (10 mM HEPES pH7.4, 150 mM NaCl, 1 mM EDTA, 0.05% Tween 20) and captured at a flow rate of 10 μl/min for 15 seconds. Data was collected for the binding of human and cynomolgus Tau monomer, each of which was injected at 0, 0.1, 0.4, 1.2, 3.7, 11.1, and 33.3 nM, using a flow rate of 30 μl/min, a contact time of 300 s, and a dissociation time of 900 s. Between cycles, the surface was regenerated with 3M magnesium chloride. Data was fit to a 1:1 binding model using BIAEvaluation software.

FIG. 26A presents affinity data for the hu37D3-H9.v76 antibody for human Tau monomer, with each curve representing a different antibody concentration. FIG. 26B presents these data for cynomolgus monkey (cyno) Tau monomer. The affinity-purified hu37D3-H9.v76, hu37D3-H9.v83, and hu37D3-H9.v93 antibodies each had a K_(D) of 0.1 nM at 37° C. for both human and cynomolgus monkey Tau monomers, as shown in Table 31. In parallel experiments, the parent hu37D3-H9.v28.A4 antibody had a K_(D) of 5-8 nM (data not shown).

TABLE 31 Affinity measurement of hu37D3 affinity-matured antibodies Human Tau monomer Cyno Tau monomer KD (nM) ka (1/Ms) kd (1/s) KD (nM) ka (1/Ms) kd (1/s) hu37D3-H9.v76 0.1 6.94 × 10⁶ 8.92 × 10⁻⁴ 0.1 9.27 × 10⁶ 1.14 × 10⁻³ hu37D3-H9.v83 0.1 6.19 × 10⁶ 8.18 × 10⁻⁴ 0.1 9.55 × 10⁶ 1.14 × 10⁻³ hu37D3-H9.v93 0.1 6.89 × 10⁶ 1.02 × 10⁻³ 0.1 1.29 × 10⁷ 1.78 × 10⁻³

Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, the descriptions and examples should not be construed as limiting the scope of the invention. The disclosures of all patent and scientific literature cited herein are expressly incorporated in their entirety by reference.

Table of Sequences SEQ ID NO Description Sequence 2 Human Tau epitope AEPRQEFEVMEDHAGTYGLGDRK (2-24) 4 Cynomolgus AEPRQEFDVMEDHAGTYGLGDRK monkey Tau epitope (2-24) 10 37D3-H9 heavy EVQLVESGGD LAKPGGSLKL SCTASGLIFR SYGMSWVRQT chain variable PDKRLEWVAT INSGGTYTYY PDSVKGRFTI SRDNAKNTLY region (VH) LQMSSLKSED TAMYYCANSY SGAMDYWGQG TSVTVSS 11 37D3-H9 light chain DDLLTQTPLS LPVSLGDPAS ISCRSSQSIV HSNGNTYFEW variable region (VL) YLQKPGQSPK LLIYKVSNRF SGVPDRFSGS GSGTDFTLKI SRVEAEDLGV YYCFQGSLVP WTFGGGTKLE IK 12 37D3-H9 HVR-H1 SYGMS 13 37D3-H9 HVR-H2 TINSGGTYTYYPDSVKG 14 37D3-H9 HVR-H3 SYSGAMDY 15 37D3-H9 HVR-L1 RSSQSIVHSNGNTYFE 16 37D3-H9 HVR-L2 KVSNRFS 17 37D3-H9 HVR-L3 FQGSLVPWT 20 37D3-H9b heavy EVQLVESGGD LAKPGGSLKL SCTASGLIFR SYGMSWVRQT chain variable PDKRLEWVAT INSGGTYTYY PDSVKGRFTI SRDNAKNTLY region (VH) LQMSSLKSED TAMYYCANSY SGAMDYWGQG TSVTVSS 21 37D3-H9b light EDLLTQTPLS LPVSLGDPAS ISCRSSQSIV HSNGNTYFEW chain variable YLQKPGQSPK LLIYKVSNRF SGVPDRFSGS GSGTDFTLKI region (VL) SRVEAEDLGV YYCFQGSLVP WTFGGGTKLE IK 22 37D3-H9b HVR-H1 SYGMS 23 37D3-H9b HVR-H2 TINSGGTYTYYPDSVKG 24 37D3-H9b HVR-H3 SYSGAMDY 25 37D3-H9b HVR-L1 RSSQSIVHSNGNTYFE 26 37D3-H9b HVR-L2 KVSNRFS 27 37D3-H9b HVR-L3 FQGSLVPWT 30 11E10-B8 heavy EVQLVESGGD LVKPGGSLKL SCAASGFTFR SYGMSWVRQT chain variable PDKRLEWVAT ISGGGSYTYY PDSVKGRFTI SRDNAKNTLY region (VH) LQMSSLKSED TAMYYCAVSY DGAMDYWGQG TSVTVSS 31 11E10-B8 light DVLMTQTPLS LPVSLGDQAS ISCRSSQSIV HSNGNTYLEW chain variable YLQKPGQSPK LLIYKVSNRF SGVPDRFSGS GSGTDFTLKI region (VL) SRVEAEDLGL YYCFQGSHVP WTFGGGTKLE IK 32 11E10-B8 HVR-H1 SYGMS 33 11E10-B8 HVR-H2 TISGGGSYTYYPDSVKG 34 11E10-B8 HVR-H3 SYDGAMDY 35 11E10-B8 HVR-L1 RSSQSIVHSNGNTYLE 36 11E10-B8 HVR-L2 KVSNRFS 37 11E10-B8 HVR-L3 FQGSHVPWT 40 54C1-H11 and EVQLVESGGD LVKPGGSLKV SCVASGFTFR SYGMSWVRQT 61E7-C4 heavy PDKRLDWVAT ISSGGNYTYY PDSVKGRFTI SRDNAKNTLY chain variable LQMSSLKSED TAMYYCASSY SGAMDYWGQG TSVTVSS region (VH) 41 54C1-H11 and DTVMTQSPLS LPVSLGDQAS ISCRSSQSIV HSNGNTYLEW 61E7-C4 light chain YLQKPGQSPK LLIYTVSNRF SGVPDRFSGS GSGTDFTLKI variable region (VL) SRVEAEDLGV YYCFQGSHVP WTFGGGTKLE IK 42 54C1-H11 and SYGMS 61E7-C4 HVR-H1 43 54C1-H11 and TISSGGNYTYYPDSVKG 61E7-C4 HVR-H2 44 54C1-H11 and SYSGAMDY 61E7-C4 HVR-H3 45 54C1-H11 and RSSQSIVHSNGNTYLE 61E7-C4 HVR-L1 46 54C1-H11 and TVSNRFS 61E7-C4 HVR-L2 47 54C1-H11 and FQGSHVPWT 61E7-C4 HVR-L3 50 3A4-H4 heavy EVQLVESGGD LVKPGGSLKL SCAASGFTFS SYGMSWVRQT chain variable PDKRLEWVAT ISSGGTYTYY PDSVKGRFTI SRDNAKNTLY region (VH) LQMSSLKSED TAMYFCATSY DGAMDYWGQG TSVTVSS 51 3A4-H4 light chain DVLMTQTPLS LPVSLGDQAS ISCRSSQNIV HSNGNTYLEW variable region (VL) YLQKPGQSPK LLIYKVSNRF SGVPDRFSGS GSGTDFTLKI SRVEAEDLGV YYCFQGTLVP WTFGGGTKLE IK 52 3A4-H4 HVR-H1 SYGMS 53 3A4-H4 HVR-H2 TISSGGTYTYYPDSVKG 54 3A4-H4 HVR-H3 SYDGAMDY 55 3A4-H4 HVR-L1 RSSQNIVHSNGNTYLE 56 3A4-H4 HVR-L2 KVSNRFS 57 3A4-H4 HVR-L3 FQGTLVPWT 60 19H6-F7 heavy EVQLVESGGD LVKPGGSLKL SCAASGFTFS SYGMSWVRQT chain variable PDKRLEWVAT ISSGGTYTYY PDSVKGRFTI SRDNAKNTLY region (VH) LQMSSLKSED TAMYYCAPSY DGAMDYWGQG TSVTVSS 61 19H6-F7 light chain DVLMTQTPLS LPVSLGDQAS ISCRSSQSIV HSNGNTYLEW variable region (VL) YLQKPGQSPK LLIYKVSNRF SGVPDRFSGS GSGTDFTLKI SRVEAEDLGV YYCFQGSLVP WTFGGGTKLE IK 62 19H6-F7 HVR-H1 SYGMS 63 19H6-F7 HVR-H2 TISSGGTYTYYPDSVKG 64 19H6-F7 HVR-H3 SYDGAMDY 65 19H6-F7 HVR-L1 RSSQSIVHSNGNTYLE 66 19H6-F7 HVR-L2 KVSNRFS 67 19H6-F7 HVR-L3 FQGSLVPWT 70 94B2-C1 heavy EVQLQQSGPE LVKPGASMKI SCKASGYSLT GYTMNWVKQS chain variable HGKNLEWIGL ISPYNGVTSY NQKFKGKATL TVDKSSNTAY region (VH) MELLSLTFED SAVYYCARQG AYWGQGTLVT VSA 71 94B2-C1 light chain DVVMTQTPLT LSVTIGQPAS ISCKSSQSLL DSDGKTYLNW variable region (VL) LLQRPGQSPK RLIYLVSKLD SGVPDRFTGS GSGTDFTLKI SRVEAEDLGV YYCWQGTHFP WTFGGGTKLE IK 72 94B2-C1 HVR-H1 GYTMN 73 94B2-C1 HVR-H2 LISPYNGVTSYNQKFKG 74 94B2-C1 HVR-H3 QGAY 75 94B2-C1 HVR-L1 KSSQSLLDSDGKTYLN 76 94B2-C1 HVR-L2 LVSKLDS 77 94B2-C1 HVR-L3 WQGTHFPWT 80 125B11-H3 heavy EVKLEESGGG LVQPGGSMKL SCVASRFIFS NYWMNWVRQS chain variable PEKGLEWVAQ IRLKSDNYAT HYAESVKGRF TISRDDSKSS region (VH) VYLQMNNLRA EDTGIYYCTG GTTYWGQGTT LTVSS 81 125B11-H3 light DIVMTQSQKF LSTSVGDRVN ITCKASQNVG TAVAWYQQKP chain variable GQSPGLLIYS ASIRYTGVPD RFTGNGSGTD FTLTISDMQS region (VL) EDLADYFCQQ FRTYPYTFGG GTKLEIK 82 125B11-H3 HVR- NYWMN H1 83 125B11-H3 HVR- QIRLKSDNYA THYAESVKG H2 84 125B11-H3 HVR- GTTY H3 85 125B11-H3 HVR- KASQNVGTAVA L1 86 125B11-H3 HVR- SASIRYT L2 87 125B11-H3 HVR- QQFRTYPYT L3 90 113F5-F7 heavy EVKLEESGGG LVQPGGSMRL SCVASEFTFS NYWMNWIRQS chain variable PEKGLEWVAQ IRLKSDNYAT HYAESVKGRF TISRDASNFS region (VH) VYLQMNNLRA EDTGIYYCTG GTSYWGQGTT LTVSS 91 113F5-F7 light DIVMTQSQKI MSTSVGDRVS ITCKASQNVG TAVAWYQQRP chain variable GHSPKLLIYS ASRRFSGVPD RFTGSGSGTD FTLTIINVQS region (VL) EDLADYFCQQ FSTYPYTFGV GTKLEIK 92 113F5-F7 HVR-H1 NYWMN 93 113F5-F7 HVR-H2 QIRLKSDNYATHYAESVKG 94 113F5-F7 HVR-H3 GTSY 95 113F5-F7 HVR-L1 KASQNVGTAVA 96 113F5-F7 HVR-L2 SASRRFS 97 113F5-F7 HVR-L3 QQFSTYPYT 100 26C1-B11 heavy EVHLQQSGAE LVRSGASVKL SCTASGFNIK DYYMYWVKQR chain variable PEQGLEWIGW IDPENGDTEY FPKFQGKATM TADTSSKTAY region (VH) LQLSSLTSED TAVYYCNAWR ARATNSALDY WGQGTSVTVS S 101 26C1-B11 light DVVMTQTPLT LSVTIGQPAS ISCKSSQSLL DSDGKTYLNW chain variable LLRRPGQSPK RLIYLVSKLD SGVPDRFTGS GSGTDFTLKI region (VL) SRVEAEDLGV YYCWQGTHFP WTFGGGTKLE IK 102 26C1-B11 HVR-H1 DYYMY 103 26C1-B11 HVR-H2 WIDPENGDTE YFPKFQG 104 26C1-B11 HVR-H3 WRARATNSAL DY 105 26C1-B11 HVR-L1 KSSQSLLDSD GKTYLN 106 26C1-B11 HVR-L2 LVSKLDS 107 26C1-B11 HVR-L3 WQGTHFPWT 110 26C1-C8 heavy EVHLQQSGAE LVRSGASVKL SCTASGFNIK DYYMYWVKQR chain variable PEQGLEWIGW IDPENGDTEY FPKFQGKATM TADTSSKTAY region (VH) LQLSSLTSED TAVYYCNAWR ARATNSALDY WGQGTSVTVS S 111 26C1-C8 light chain DVVMTQTPLT LSVTIGQPAS ISCKSSQSLL DSDGKTYLNW variable region (VL) LLRRPGQSPK RLIYLVSKLD SGVPDRFTGS GSGTDFTLKI SRVEAEDLGV YYCWQGTHFP WTFGGGTKLE IK 112 26C1-C8 HVR-H1 DYYMY 113 26C1-C8 HVR-H2 WIDPENGDTE YFPKFQG 114 26C1-C8 HVR-H3 WRARATNSAL DY 115 26C1-C8 HVR-L1 KSSQSLLDSD GKTYLN 116 26C1-C8 HVR-L2 LVSKLDS 117 26C1-C8 HVR-L3 WQGTHFPWT 120 30G1-B2 heavy QVQLQQSGAE LVRPGASVTL SCKASGYTFT DYEMYWVKQT chain variable PVHGLEWIGA IDPETGDTAY NQKFKGKATL TADKSSNTAY region (VH) MELRSLTSED SAVYYCIRQY GNWFPYWGQG TLVTVSA 121 30G1-B2 light chain DVVMTQTPLS LPVSLGDQAS ISCRSSQSLV HANGNTYLHW variable region (VL) FLQKPGLSPK LLIYKVSNRF SGVPDRFSGG GSGTDFTLKI TRLEAEDLGV YFCSQSTHVP FTFGSGTKLE IK 122 30G1-B2 HVR-H1 DYEMY 123 30G1-B2 HVR-H2 AIDPETGDTAYNQKFKG 124 30G1-B2 HVR-H3 QYGNWFPY 125 30G1-B2 HVR-L1 RSSQSLVHANGNTYLH 126 30G1-B2 HVR-L2 KVSNRFS 127 30G1-B2 HVR-L3 SQSTHVPFT 130 66F5-A1 heavy QVQLQQSGAE LVRPGASVTL SCKASGYTFI DYEMNWVKQT chain variable PVHGLEWIGA IDPENGGTAY NQKFKGKAIV TADKSSSTAY region (VH) MELRSLTSED SAVYYCSGPH FDYWGQGTTL TVSS 131 66F5-A1 light chain DIVMTQSPSS LAMSVGQKVT MSCKSSQSLL NSSTQKNYLA variable region (VL) WYQQKPGQSP KLLVYFASTR ESGVPDRFIG SGSGTDFTLT ISSVQAEDLA DYFCQQHYST PYTFGGGTKL EIK 132 66F5-A1 HVR-H1 DYEMN 133 66F5-A1 HVR-H2 AIDPENGGTA YNQKFKG 134 66F5-A1 HVR-H3 PHFDY 135 66F5-A1 HVR-L1 KSSQSLLNSS TQKNYLA 136 66F5-A1 HVR-L2 FASTRES 137 66F5-A1 HVR-L3 QQHYSTPYT 140 123E9-A1 heavy EVQLQQSGPE LVKPGASVKM SCKASGYTFT DYYMKWVKQS chain variable HGKSLEWIGD IDPNNGGTSY NQKFKGKATL TVDKSSSTAY region (VH) MQLNSLTSED SAVYYCARSA GFGDSFSFWG LGTLVTVSA 141 123E9-A1 light DVLMTQTPLS LPVSLGDQAS ISCRSSQSIV HSNGNTYLEW chain variable YLQKPGQSPK LLIYKVSNRF SGVPDRFSGS GSGTDFTLKI region (VL) SRVEAEDLGF YYCFQGSHVP PTFGGGTKLE IK 142 123E9-A1 HVR-H1 DYYMK 143 123E9-A1 HVR-H2 DIDPNNGGTSYNQKFKG 144 123E9-A1 HVR-H3 SAGFGDSFSF 145 123E9-A1 HVR-L1 RSSQSIVHSNGNTYLE 146 123E9-A1 HVR-L2 KVSNRFS 147 123E9-A1 HVR-L3 FQGSHVPPT 150 15C6-A7 heavy EVQLQQSGPE LVKPGASVMM TCKASGYTFT DYYMKWVKQS chain variable NGKSLEWIGD LDPYTGGANY NQKFKGKATL TVDKSSSTAY region (VH) MHLNSLTSED SAVYYCARSR GYGDSFAYWG QGTLVTVSA 151 15C6-A7 light chain DVLMTQTPLS LPVSLGDQAS ISCRSSQNIV HSNGNTYLEW variable region (VL) YLQKPGQSPK LLIYKVSNRF SGVPDKFSGS GSGTDFTLKI SRVEAEDLGV YFCFQGSHVP PTFGGGTKLE IK 152 15C6-A7 HVR-H1 DYYMK 153 15C6-A7 HVR-H2 DLDPYTGGAN YNQKFKG 154 15C6-A7 HVR-H3 SRGYGDSFAY 155 15C6-A7 HVR-L1 RSSQNIVHSN GNTYLE 156 15C6-A7 HVR-L2 KVSNRFS 157 15C6-A7 HVR-L3 FQGSHVPPT 160 19F8-B1 heavy EVQLQQSGPE LVKPGASVKM SCKASGYTFT DYYMKWVKQS chain variable HGKSLEWIGD LNPNNGGTLY NQKFKGQATL TVDKSSSTAY region (VH) MQFNSLTSED SAVYYCARSA GYGDSFAYWG QGTLVTVSA 161 19F8-B1 light chain DVLMTQTPLS LPVSLGDQAS ISCRSSQNIV HSNGNTYLEW variable region (VL) YLQKPGQSPK LLIYKVSNRF SGVPDRFSGS GSGTDFTLKI SRVEAEDLGI YFCFQGSHVP PTFGGGTKLE IK 162 19F8-B1 HVR-H1 DYYMK 163 19F8-B1 HVR-H2 DLNPNNGGTL YNQKFKG 164 19F8-B1 HVR-H3 SAGYGDSFAY 165 19F8-B1 HVR-L1 RSSQNIVHSN GNTYLE 166 19F8-B1 HVR-L2 KVSNRFS 167 19F8-B1 HVR-L3 FQGSHVPPT 170 24A11-D5 heavy EVQLQQSGPE LVKPGASVKM SCKASGYTFT DYYMKWVKQS chain variable HGKSLEWIGD LNPKNGGIIY NQKFKGQATL TVDKSSSTAY region (VH) MQLNSLTSED SAVFYCARSG GYGDSFAYWG QGTLVTVSA 171 24A11-D5 light DVLMTQTPLS LPVSLGDQAS ISCRSSQNIV HSNGNTYLEW chain variable YLQKPGQSPK LLIYKVSNRF SGVPDRFSGS GSGTDFTLKI region (VL) SRVEAEDLGI YFCFQGSHVP PTFGGGTKLE IK 172 24A11-D5 HVR-H1 DYYMK 173 24A11-D5 HVR-H2 DLNPKNGGII YNQKFKG 174 24A11-D5 HVR-H3 SGGYGDSFAY 175 24A11-D5 HVR-L1 RSSQNIVHSN GNTYLE 176 24A11-D5 HVR-L2 KVSNRFS 177 24A11-D5 HVR-L3 FQGSHVPPT 180 126F11-G11 heavy EVQLQQSGAE LVRPGASVKL SCTASGFNIK DDYMHWVKQR chain variable PEQGLEWIGW IDPENGDTEY ASKFQGKATI TTDTSSNTAY region (VH) LQLSSLTSED TAVYYCLDFA YGYWGQGTTL TVSS 181 126F11-G11 light DVLMTQTPLS LPVSLGDQAS ISCRSSQSIV HSNGNTYLEW chain variable YLQKPGQSPK LLIYKVSNRF SGVPDRFSGS GSGTDFTLKI region (VL) SRVEAEDLGV YYCFQGSHVP PAFGGGTKLE IK 182 126F11-G11 HVR- DDYMH H1 183 126F11-G11 HVR- WIDPENGDTE YASKFQG H2 184 126F11-G11 HVR- FAYGY H3 185 126F11-G11 HVR- RSSQSIVHSN GNTYLE L1 186 126F11-G11 HVR- KVSNRFS L2 187 126F11-G11 HVR- FQGSHVPPA L3 190 89F4-A1 heavy EVQLVESGGG LVQPKGSLKL SCAASGFTFN TYAMNWVRQA chain variable PGKGLEWVAR IRSKSNNYAA YFADSVKDRF TISRDDSQTM region (VH) LYLQMNNLKS EDTAMYYCVS GGNYVPFAYW GQGTLVTVSA 191 89F4-A1 light chain NIMMTQSPSS LAVSAGEKVT MSCKSSQSVF YSSEQRNYLA variable region (VL) WYQQKPGQSP KLLISWASTR ESGVPDRFTG SGSGTDFTLT ISSVQGEDLA VYYCHQYLSS FTFGSGTKLE IK 192 89F4-A1 HVR-H1 TYAMN 193 89F4-A1 HVR-H2 RIRSKSNNYA AYFADSVKD 194 89F4-A1 HVR-H3 GGNYVPFAY 195 89F4-A1 HVR-L1 KSSQSVFYSS EQRNYLA 196 89F4-A1 HVR-L2 WASTRES 197 89F4-A1 HVR-L3 HQYLSSFT 200 93A8-D2 heavy EVQLQQSGPV LVKPGASVKM SCKASGYTFT DYYVNWVKQS chain variable HGKGLEWIGL INPNNGRTSY NQNFNDKATL TVDKSSSTAF region (VH) MDLNSLTSED SAVYYCTREG GTGYWGQGTT LSVSS 201 93A8-D2 light chain DVVMTQTPLT LSVTIGQPAS ISCKSSQSLL DSDGKTYLNW variable region (VL) LLQRPGQSPR RLIYLVSKLD SGVPDRFTGS GSGTDFTLKI SRVAAEDLGV YYCWQGTHFP RTFGGGTKLE IK 202 93A8-D2 HVR-H1 DYYVN 203 93A8-D2 HVR-H2 LINPNNGRTSYNQNFND 204 93A8-D2 HVR-H3 EGGTGY 205 93A8-D2 HVR-L1 KSSQSLLDSDGKTYLN 206 93A8-D2 HVR-L2 LVSKLDS 207 93A8-D2 HVR-L3 WQGTHFPRT 210 14F5-D9 heavy EVKLVESGGG LVQPGGSLRL SCATSGFTFS DFYMEWVRQS chain variable PGKRLEWIAA SKNKANDYTT EYNASVKDRF FVSRDTSQSI region (VH) LYLQMNALRA EDTAIYYCAR DALGTVFAYW GQGTLVTVSA 211 14F5-D9 light chain DVVMTQTPLS LPVSLGDQAS ISCRSSQSLV HSNGNTYLHW variable region (VL) YLQKPGQSPK LLIYKVFNRF SGVPDRFSGS GSGTDFTLKI SRVEAEDLGV YFCSQSTLVP LTFGAGTKLE LK 212 14F5-D9 HVR-H1 DFYME 213 14F5-D9 HVR-H2 ASKNKANDYT TEYNASVKD 214 14F5-D9 HVR-H3 DALGTVFAY 215 14F5-D9 HVR-L1 RSSQSLVHSN GNTYLH 216 14F5-D9 HVR-L2 KVFNRFS 217 14F5-D9 HVR-L3 SQSTLVPLT 220 73H6-B8 heavy QVQLKESGPG LVAPSQSLSI TCTISGFSLT SYGVHWVRQP chain variable PGKGLEWLVV IWSDGSTTYN SALKSRLSIS KDNSKSQVFL region (VH) KMNSLQTDDT AMYYCARQGG FITTAYYAMD YWGQGTSVTV SS 221 73H6-B8 light chain DIVMSQSPSS LAVSAGEKVT MSCKSSQSLL NSRTRKNYLA variable region (VL) WYQQKPGQSP KLLIYWASTR ESGVPDRFTG SGSGTDFTLT ISSVQAEDLA VYYCKQSYNL YTFGGGTKLE IK 222 73H6-B8 HVR-H1 SYGVH 223 73H6-B8 HVR-H2 VIWSDGSTTY NSALKS 224 73H6-B8 HVR-H3 QGGFITTAYY AMDY 225 73H6-B8 HVR-L1 KSSQSLLNSR TRKNYLA 226 73H6-B8 HVR-L2 WASTRES 227 73H6-B8 HVR-L3 KQSYNLYT 230 22G7-C9 heavy QIQLVQSGPE LKKPGETVKI SCKASGYTFT DCSIHWVKQA chain variable PGEGLKWMGW INTETGEPSY ADDFKGRFAF SLETSASTAF region (VH) LQINNLKSED TASYFCGTAY YRYDGALDYW GQGTSVTVSS 231 22G7-C9 light chain DIVLTQSPAS LAVSLGQRAT ISCRASQSVS TSSYSYMHWF variable region (VL) QQKPGQPPKL LIKYASNLES GVPARFSGSG SGTDFTLNIH PVEEEDTATY YCQHSWELPW TFGGGTKLEI K 232 22G7-C9 HVR-H1 DCSIH 233 22G7-C9 HVR-H2 WINTETGEPS YADDFKG 234 22G7-C9 HVR-H3 AYYRYDGALD Y 235 22G7-C9 HVR-L1 RASQSVSTSS YSYMH 236 22G7-C9 HVR-L2 YASNLES 237 22G7-C9 HVR-L3 QHSWELPWT 240 7A11-C12 heavy QIQLVQSGPD LKKPGETVKI SCKASGYTFT NYGMNWVKQA chain variable PGKGLKWMGW INTNTGEPTY AEEFKGRFAF SLETSASTAY region (VH) LQIDNLKNED TATYFCARGT VSFPYWGQGT LVTVSA 241 7A11-C12 light DVVMSQTPLS LPVSLGDHAS ISCRSSQNLV HSDGNTYLHW chain variable YLQKPGQSPK LLIYKVSNRF SGVPDRFSGS GSGTDFTLKI region (VL) SRVEAEDLGV YFCSQSTHVI FTFGSGTKLE IK 242 7A11-C12 HVR-H1 NYGMN 243 7A11-C12 HVR-H2 WINTNTGEPT YAEEFKG 244 7A11-C12 HVR-H3 GTVSFPY 245 7A11-C12 HVR-L1 RSSQNLVHSD GNTYLH 246 7A11-C12 HVR-L2 KVSNRFS 247 7A11-C12 HVR-L3 SQSTHVIFT 250 12A10-E8 heavy QIQLVQSGPE LKKPGETVKI SCKASGYTFT NYGMNWVKQA chain variable PGKGLKWMGW INMYTGEPTY GDDFKGRFVF SLETSVSTVY region (VH) LQINNLKKED TATFFCARGG RPDYWGQGTS VTVSS 251 12A10-E8 light DVLMTQTPLS LPVSLGDQAS ISCRSSQSIV HSNGNTYLEW chain variable YLQKPGQSPK LLIYKVFNRF SGVPDRFSGS GSGTDFTLKI region (VL) NRVEAEDLGV YYCLQGSHVP YTFGGGTKLE IK 252 12A10-E8 HVR-H1 NYGMN 253 12A10-E8 HVR-H2 WINMYTGEPT YGDDFKG 254 12A10-E8 HVR-H3 GGRPDY 255 12A10-E8 HVR-L1 RSSQSIVHSN GNTYLE 256 12A10-E8 HVR-L2 KVFNRFS 257 12A10-E8 HVR-L3 LQGSHVPYT 260 55E7-F11 heavy EVKLEESGGG LVQPGGSMKL SCVASGFTFS NYWMNWVRQS chain variable PEKGLEWVAQ IRLKSDNYAT HYAESVKGRF TISRDDSKSS region (VH) VYLQMNNLRA EDTGIYYCAG YFYGGYFDVW GTGTTVTVSS 261 55E7-F11 light ELVLTQSPTT MAASPGKKIT ITCSASSSIS SNYLHWYQQK chain variable PGFSPKLLIY RTSNLASGVP ARFSGSGSGT SYSLTIGTME region (VL) AEDVATYYCQ QGSSLPFTFG SGTKLEIK 262 55E7-F11 HVR-H1 NYWMN 263 55E7-F11 HVR-H2 QIRLKSDNYA THYAESVKG 264 55E7-F11 HVR-H3 YFYGGYFDV 265 55E7-F11 HVR-L1 SASSSISSNY LH 266 55E7-F11 HVR-L2 RTSNLAS 267 55E7-F11 HVR-L3 QQGSSLPFT 270 52F6-F11 heavy QVQLQQSGTE LAKPGASVKL SCKASGYTFT HYWMHWIKQR chain variable PGQGLEWIGY IYPTNDYTKY NQNFRDKATL TADESSNSAY region (VH) MQLNSLTYED SAVYYCARAG NRVFDFWGQG TTLTVSS 271 52F6-F11 light QAVVTQESAL TTSPGETVTL TCRSSTGAVT TSNFANWVQE chain variable KPDHLFTGLI GGTNNRAPGV PARFSGSLIG DKAALTITGA region (VL) QTEDEAIYFC ALWYSNLWVF GGGTKLTVL 272 52F6-F11 HVR-H1 HYWMH 273 52F6-F11 HVR-H2 YIYPTNDYTK YNQNFRD 274 52F6-F11 HVR-H3 AGNRVFDF 275 52F6-F11 HVR-L1 RSSTGAVTTS NFAN 276 52F6-F11 HVR-L2 GTNNRAP 277 52F6-F11 HVR-L3 ALWYSNLWV 280 Hu37D3-H9.v1 EVQLVESGGG LVQPGGSLRL SCAASGLIFR SYGMSWVRQA heavy chain variable PGKGLEWVAT INSGGTYTYY PDSVKGRFTI SRDNSKNTLY region (VH) LQMNSLRAED TAVYYCANSY SGAMDYWGQG TLVTVSS 281 Hu37D3-H9.v1 EDQLTQSPSS LSASVGDRVT ITCRSSQSIV HSNGNTYFEW light chain variable YQQKPGKSPK LLIYKVSNRF SGVPSRFSGS GSGTDFTLTI region (VL) SSLQPEDFAT YYCFQGSLVP WTFGQGTKVE IK 282 Hu37D3-H9.v1 SYGMS HVR-H1 283 Hu37D3-H9.v1 TINSGGTYTYYPDSVKG HVR-H2 284 Hu37D3-H9.v1 SYSGAMDY HVR-H3 285 Hu37D3-H9.v1 RSSQSIVHSNGNTYFE HVR-L1 286 Hu37D3-H9.v1 KVSNRFS HVR-L2 287 Hu37D3-H9.v1 FQGSLVPWT HVR-L3 288 Hu37D3-H9.v1 EVQLVESGGG LVQPGGSLRL SCAASGLIFR SYGMSWVRQA IgG1 heavy chain PGKGLEWVAT INSGGTYTYY PDSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCANSY SGAMDYWGQG TLVTVSSAST KGPSVFPLAP SSKSTSGGTA ALGCLVKDYF PEPVTVSWNS GALTSGVHTF PAVLQSSGLY SLSSVVTVPS SSLGTQTYIC NVNHKPSNTK VDKKVEPKSC DKTHTCPPCP APELLGGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSHED PEVKFNWYVD GVEVHNAKTK PREEQYNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKALPA PIEKTISKAK GQPREPQVYT LPPSREEMTK NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSKL TVDKSRWQQG NVFSCSVMHE ALHNHYTQKS LSLSPGK 289 Hu37D3-H9.v1 EDQLTQSPSS LSASVGDRVT ITCRSSQSIV HSNGNTYFEW IgG1 light chain YQQKPGKSPK LLIYKVSNRF SGVPSRFSGS GSGTDFTLTI SSLQPEDFAT YYCFQGSLVP WTFGQGTKVE IKRTVAAPSV FIFPPSDEQL KSGTASVVCL LNNFYPREAK VQWKVDNALQ SGNSQESVTE QDSKDSTYSL SSTLTLSKAD YEKHKVYACE VTHQGLSSPV TKSFNRGEC 290 Hu37D3-H9.v5 EVQLVESGGG LVQPGGSLRL SCAASGLIFR SYGMSWVRQA heavy chain variable PGKGLEWVAT INSGGTYTYY PDSVKGRFTI SRDNSKNTLY region (VH) LQMNSLRAED TAVYYCANSY SGAMDYWGQG TLVTVSS 291 Hu37D3-H9.v5 EDVLTQTPLS LPVTPGQPAS ISCRSSQSIV HSNGNTYFEW light chain variable YLQKPGQSPQ LLIYKVSNRF SGVPDRFSGS GSGTDFTLKI region (VL) SRVEAEDVGV YYCFQGSLVP WTFGQGTKVE IK 292 Hu37D3-H9.v5 SYGMS HVR-H1 293 Hu37D3-H9.v5 TINSGGTYTYYPDSVKG HVR-H2 294 Hu37D3-H9.v5 SYSGAMDY HVR-H3 295 Hu37D3-H9.v5 RSSQSIVHSNGNTYFE HVR-L1 296 Hu37D3-H9.v5 KVSNRFS HVR-L2 297 Hu37D3-H9.v5 FQGSLVPWT HVR-L3 300 Hu94B2.v105 heavy EVQLVQSGAE VKKPGASVKV SCKASGYSLT GYTMNWVRQA chain variable PGQGLEWIGL ISPYNGVTSY NQKFKGRATL TVDKSTSTAY region (VH) LELSSLRSED TAVYYCARQG AYWGQGTLVT VSS 301 Hu94B2.v105 light DIVMTQTPLS LPVTPGQPAS ISCKSSQSLL DSDGKTYLNW chain variable LLQKPGQSPQ RLIYLVSKLD SGVPDRFSGS GSGTDFTLKI region (VL) SRVEAEDVGV YYCWQGTHFP WTFGQGTKVE IK 302 Hu94B2.v105 GYTMN HVR-H1 303 Hu94B2.v105 LISPYNGVTSYNQKFKG HVR-H2 304 Hu94B2.v105 QGAY HVR-H3 305 Hu94B2.v105 KSSQSLLDSDGKTYLN HVR-L1 306 Hu94B2.v105 LVSKLDS HVR-L2 307 Hu94B2.v105 WQGTHFPWT HVR-L3 310 hu125B11.v17 EVQLVESGGG LVQPGGSLRL SCAASRFIFS NYWMNWVRQA heavy chain PGKGLEWVAQ IRLKSDNYAT HYAESVKGRF TISRDDSKNT variable region LYLQMNSLRA EDTAVYYCTG GTTYWGQGTL VTVSS (VH) 311 hu125B11.v17 DIQMTQSPSS LSASVGDRVT ITCKASQNVG TAVAWYQQKP light chain GKSPKLLIYS ASIRYTGVPS RFSGSGSGTD FTLTISSLQP variable region EDFATYFCQQ FRTYPYTFGQ GTKVEIK (VL) 312 hu125B11.v17 NYWMN HVR-H1 313 hu125B11.v17 QIRLKSDNYATHYAESVKG HVR-H2 314 hu125B11.v17 GTTY HVR-H3 315 hu125B11.v17 KASQNVGTAVA HVR-L1 316 hu125B11.v17 SASIRYT HVR-L2 317 hu125B11.v17 QQFRTYPYT HVR-L3 320 hu125B11.v26 EVQLVESGGG LVQPGGSLRL SCAASRFIFS NYWMNWVRQA heavy chain PGKGLEWVAQ IRLKSDNYAT HYAESVKGRF TISRDNSKNT variable region LYLQMNSLRA EDTAVYYCTG GTTYWGQGTL VTVSS (VH) 321 hu125B11.v26 DIQMTQSPSS LSASVGDRVT ITCKASQNVG TAVAWYQQKP light chain GKAPKLLIYS ASIRYTGVPS RFSGSGSGTD FTLTISSLQP variable region EDFATYFCQQ FRTYPYTFGQ GTKVEIK (VL) 322 hu125B11.v26 NYWMN HVR-H1 323 hu125B11.v26 QIRLKSDNYATHYAESVKG HVR-H2 324 hu125B11.v26 GTTY HVR-H3 325 hu125B11.v26 KASQNVGTAVA HVR-L1 326 hu125B11.v26 SASIRYT HVR-L2 327 hu125B11.v26 QQFRTYPYT HVR-L3 330 hu125B11.v28 EVQLVESGGG LVQPGGSLRL SCAASRFIFS NYWMNWVRQA heavy chain PGKGLEWVAQ IRLKSDNYAT HYAESVKGRF TISRDNSKNT variable region LYLQMNSLRA EDTAVYYCTG GTTYWGQGTL VTVSS (VH) 331 hu125B11.v28 DIQMTQSPSS LSASVGDRVT ITCKASQNVG TAVAWYQQKP light chain GKAPKLLIYS ASIRYTGVPS RFSGSGSGTD FTLTISSLQP variable region EDFATYYCQQ FRTYPYTFGQ GTKVEIK (VL) 332 hu125B11.v28 NYWMN HVR-H1 333 hu125B11.v28 QIRLKSDNYATHYAESVKG HVR-H2 334 hu125B11.v28 GTTY HVR-H3 335 hu125B11.v28 KASQNVGTAVA HVR-L1 336 hu125B11.v28 SASIRYT HVR-L2 337 hu125B11.v28 QQFRTYPYT HVR-L3 340 Hu37D3-H9.v28.A4 EVQLVESGGG LVQPGGSLRL SCAASGLIFR SYGMSWVRQA heavy chain variable PGKGLEWVAT INSGGTYTYY PDSVKGRFTI SRDNSKNTLY region (VH) LQMNSLRAED TAVYYCANSY SGAMDYWGQG TLVTVSS 341 Hu37D3-H9.v28.A4 DDVLTQTPLS LPVTPGQPAS ISCRSSQSIV HSNGNTYLEW light chain variable YLQKPGQSPQ LLIYKVSNRF SGVPDRFSGS GSGTDFTLKI region (VL) SRVEAEDVGV YYCFQGSLVP WTFGQGTKVE IK 342 Hu37D3-H9.v28.A4 SYGMS HVR-H1 343 Hu37D3-H9.v28.A4 TINSGGTYTYYPDSVKG HVR-H2 344 Hu37D3-H9.v28.A4 SYSGAMDY HVR-H3 345 Hu37D3-H9.v28.A4 RSSQSIVHSNGNTYLE HVR-L1 346 Hu37D3-H9.v28.A4 KVSNRFS HVR-L2 347 Hu37D3-H9.v28.A4 FQGSLVPWT HVR-L3 348 Hu37D3-H9.v28.A4 EVQLVESGGG LVQPGGSLRL SCAASGLIFR SYGMSWVRQA IgG4-S228P.YTE PGKGLEWVAT INSGGTYTYY PDSVKGRFTI SRDNSKNTLY heavy chain LQMNSLRAED TAVYYCANSY SGAMDYWGQG TLVTVSSAST KGPSVFPLAP CSRSTSESTA ALGCLVKDYF PEPVTVSWNS GALTSGVHTF PAVLQSSGLY SLSSWTVPS SSLGTKTYTC NVDHKPSNTK VDKRVESKYG PPCPPCPAPE FLGGPSVFLF PPKPKDTLYI TREPEVTCVV VDVSQEDPEV QFNWYVDGVE VHNAKTKPRE EQFNSTYRW SVLTVLHQDW LNGKEYKCKV SNKGLPSSIE KTISKAKGQP REPQVYTLPP SQEEMTKNQV SLTCLVKGFY PSDIAVEWES NGQPENNYKT TPPVLDSDGS FFLYSRLTVD KSRWQEGNVF SCSVMHEALH NHYTQKSLSL SLGK 602 Hu37D3-H9.v28.A4 EVQLVESGGG LVQPGGSLRL SCAASGLIFR SYGMSWVRQA IgG4-S228P.YTE PGKGLEWVAT INSGGTYTYY PDSVKGRFTI SRDNSKNTLY des-K heavy chain LQMNSLRAED TAVYYCANSY SGAMDYWGQG TLVTVSSAST KGPSVFPLAP CSRSTSESTA ALGCLVKDYF PEPVTVSWNS GALTSGVHTF PAVLQSSGLY SLSSVVTVPS SSLGTKTYTC NVDHKPSNTK VDKRVESKYG PPCPPCPAPE FLGGPSVFLF PPKPKDTLYI TREPEVTCVV VDVSQEDPEV QFNWYVDGVE VHNAKTKPRE EQFNSTYRVV SVLTVLHQDW LNGKEYKCKV SNKGLPSSIE KTISKAKGQP REPQVYTLPP SQEEMTKNQV SLTCLVKGFY PSDIAVEWES NGQPENNYKT TPPVLDSDGS FFLYSRLTVD KSRWQEGNVF SCSVMHEALH NHYTQKSLSL SLG 349 Hu37D3-H9.v28.A4 DDVLTQTPLS LPVTPGQPAS ISCRSSQSIV HSNGNTYLEW IgG4-S228P.YTE YLQKPGQSPQ LLIYKVSNRF SGVPDRFSGS GSGTDFTLKI light chain SRVEAEDVGV YYCFQGSLVP WTFGQGTKVE IKRTVAAPSV FIFPPSDEQL KSGTASVVCL LNNFYPREAK VQWKVDNALQ SGNSQESVTE QDSKDSTYSL SSTLTLSKAD YEKHKVYACE VTHQGLSSPV TKSFNRGEC 442 hu125B11-H3.LC1 DIQMTQSPSS LSASVGDRVT ITCKASQNVG TAVAWYQQKP GKSPKLLIYS ASIRYTGVPS RFSGSGSGTD FTLTISSLQP EDFATYFCQQ FRTYPYTFGQ GTKVEIK 443 hu125B11-H3.LC2 DIQMTQSPSS LSASVGDRVT ITCKASQNVG TAVAWYQQKP GKAPKLLIYS ASIRYTGVPS RFSGSGSGTD FTLTISSLQP EDFATYFCQQ FRTYPYTFGQ GTKVEIK 444 hu125B11-H3.LC3 DIQMTQSPSS LSASVGDRVT ITCKASQNVG TAVAWYQQKP GKSPKLLIYS ASIRYTGVPS RFSGSGSGTD FTLTISSLQP EDFATYYCQQ FRTYPYTFGQ GTKVEIK 445 hu125B11-H3.LC4 DIQMTQSPSS LSASVGDRVT ITCKASQNVG TAVAWYQQKP GKAPKLLIYS ASIRYTGVPS RFSGSGSGTD FTLTISSLQP EDFATYYCQQ FRTYPYTFGQ GTKVEIK 446 hu125B11-H3.HC1 EVQLVESGGG LVQPGGSLRL SCAASRFIFS NYWMNWVRQA PGKGLEWVAQ IRLKSDNYAT HYAESVKGRF TISRDDSKNT VYLQMNSLRA EDTAVYYCTG GTTYWGQGTL VTVSS 447 hu125B11-H3.HC2 EVQLVESGGG LVQPGGSLRL SCAASRFIFS NYWMNWVRQA PGKGLEWVAQ IRLKSDNYAT HYAESVKGRF TISRDNSKNT VYLQMNSLRA EDTAVYYCTG GTTYWGQGTL VTVSS 448 hu125B11-H3.HC3 EVQLVESGGG LVQPGGSLRL SCAASRFIFS NYWMNWVRQA PGKGLEWVAQ IRLKSDNYAT HYAESVKGRF TISRDDSKNT LYLQMNSLRA EDTAVYYCTG GTTYWGQGTL VTVSS 449 hu125B11-H3.HC4 EVQLVESGGG LVQPGGSLRL SCAASRFIFS NYWMNWVRQA PGKGLEWVAQ IRLKSDNYAT HYAESVKGRF TISRDNSKNT LYLQMNSLRA EDTAVYYCTG GTTYWGQGTL VTVSS 450 hu125B11-H3.HC5 EVQLVESGGG LVQPGGSLRL SCAASRFIFS NYYMNWVRQA PGKGLEWVAQ IRLKSDNYAT HYAESVKGRF TISRDDSKNT VYLQMNSLRA EDTAVYYCTG GTTYWGQGTL VTVSS 451 hu125B11-H3.HC6 EVQLVESGGG LVQPGGSLRL SCAASRFIFS NYFMNWVRQA PGKGLEWVAQ IRLKSDNYAT HYAESVKGRF TISRDDSKNT VYLQMNSLRA EDTAVYYCTG GTTYWGQGTL VTVSS 452 Hu94B2.HC1 EVQLVQSGAE VKKPGASVKV SCKASGYSLT GYTMNWVRQA PGQGLEWIGL ISPYNGVTSY NQKFKGRATL TVDKSTSTAY LELSSLRSED TAVYYCARQG AYWGQGTLVT VSS 453 Hu94B2.HC2 EVQLVQSGAE VKKPGASVKV SCKASGYSLT GYTMNWVRQA PGQGLEWIGL ISPYNGVTSY NQKFKGRVTL TVDKSTSTAY LELSSLRSED TAVYYCARQG AYWGQGTLVT VSS 454 Hu94B2.HC3 EVQLVQSGAE VKKPGASVKV SCKASGYSLT GYTMNWVRQA PGQGLEWIGL ISPYNGVTSY NQKFKGRATI TVDKSTSTAY LELSSLRSED TAVYYCARQG AYWGQGTLVT VSS 455 Hu94B2.HC4 EVQLVQSGAE VKKPGASVKV SCKASGYSLT GYTMNWVRQA PGQGLEWIGL ISPYNGVTSY NQKFKGRATL TRDKSTSTAY LELSSLRSED TAVYYCARQG AYWGQGTLVT VSS 456 Hu94B2.HC5 EVQLVQSGAE VKKPGASVKV SCKASGYSLT GYTMNWVRQA PGQGLEWIGL ISPYNGVTSY NQKFKGRATL TVDTSTSTAY LELSSLRSED TAVYYCARQG AYWGQGTLVT VSS 457 Hu94B2.HC6 EVQLVQSGAE VKKPGASVKV SCKASGYSLT GYTMNWVRQA PGQGLEWIGL ISPYNGVTSY NQKFKGRVTI TVDKSTSTAY LELSSLRSED TAVYYCARQG AYWGQGTLVT VSS 458 Hu94B2.HC7 EVQLVQSGAE VKKPGASVKV SCKASGYSLT GYTMNWVRQA PGQGLEWIGL ISPYNGVTSY NQKFKGRVTI TRDKSTSTAY LELSSLRSED TAVYYCARQG AYWGQGTLVT VSS 459 Hu94B2.HC8 EVQLVQSGAE VKKPGASVKV SCKASGYSLT GYTMNWVRQA PGQGLEWIGL ISPYNGVTSY NQKFKGRVTI TVDTSTSTAY LELSSLRSED TAVYYCARQG AYWGQGTLVT VSS 460 Hu94B2.LC9 DVVMTQTPLS LPVTPGQPAS ISCKSSQSLL DSDGKTYLNW LLQKPGQSPQ RLIYLVSKLD SGVPDRFSGS GSGTDFTLKI SRVEAEDVGV YYCWQGTHFP WTFGQGTKVE IK 461 Hu94B2.LC10 DVVMTQTPLS LPVTPGQPAS ISCKSSQSLL DSDGKTYLNW LLQKPGQSPQ LLIYLVSKLD SGVPDRFSGS GSGTDFTLKI SRVEAEDVGV YYCWQGTHFP WTFGQGTKVE IK 462 Hu94B2.LC11 DVVMTQTPLS LPVTPGQPAS ISCKSSQSLL DSDGKTYLNW YLQKPGQSPQ RLIYLVSKLD SGVPDRFSGS GSGTDFTLKI SRVEAEDVGV YYCWQGTHFP WTFGQGTKVE IK 463 Hu94B2.LC12 DVVMTQTPLS LPVTPGQPAS ISCKSSQSLL DSDGKTYLNW YLQKPGQSPQ LLIYLVSKLD SGVPDRFSGS GSGTDFTLKI SRVEAEDVGV YYCWQGTHFP WTFGQGTKVE IK 464 Hu94B2.LC13 DIVMTQTPLS LPVTPGQPAS ISCKSSQSLL DSDGKTYLNW LLQKPGQSPQ RLIYLVSKLD SGVPDRFSGS GSGTDFTLKI SRVEAEDVGV YYCWQGTHFP WTFGQGTKVE IK 465 Hu94B2.LC14 DIVMTQTPLS LPVTPGQPAS ISCKSSQSLL DSDGKTYLNW LLQKPGQSPQ LLIYLVSKLD SGVPDRFSGS GSGTDFTLKI SRVEAEDVGV YYCWQGTHFP WTFGQGTKVE IK 466 Hu94B2.LC15 DIVMTQTPLS LPVTPGQPAS ISCKSSQSLL DSDGKTYLNW YLQKPGQSPQ RLIYLVSKLD SGVPDRFSGS GSGTDFTLKI SRVEAEDVGV YYCWQGTHFP WTFGQGTKVE IK 467 Hu94B2.LC16 DIVMTQTPLS LPVTPGQPAS ISCKSSQSLL DSDGKTYLNW YLQKPGQSPQ LLIYLVSKLD SGVPDRFSGS GSGTDFTLKI SRVEAEDVGV YYCWQGTHFP WTFGQGTKVE IK 468 Hu37D3-H9.v5.1 RSSQSIVHSNANTYFE HVR-L1 469 Hu37D3-H9.v5.2 RSSQSIVHSSGNTYFE HVR-L1 470 Hu37D3-H9.v5.3 RSSQSIVHSDGNTYFE HVR-L1 471 Hu37D3-H9.v5.4 RSSQSIVHSQGNTYFE HVR-L1 472 Hu37D3-H9.v5.5 RSSQSIVHSEGNTYFE HVR-L1 473 Hu37D3-H9.v5.6 RSSQSIVHSAGNTYFE HVR-L1 474 Hu37D3-H9.v5.7 RSSQSIVHSNGDTYFE HVR-L1 475 Hu37D3-H9.v5.8 RSSQSIVHSNGQTYFE HVR-L1 476 Hu37D3-H9.v5.9 RSSQSIVHSNGETYFE HVR-L1 477 Hu37D3-H9.v5.10 RSSQSIVHSNGATYFE HVR-L1 478 Hu37D3-H9.v5.11 RSSQSIVHSNGSTYFE HVR-L1 479 Hu37D3.v28 HVR- RSSQSIVHSNGNTYFE L1 480 Hu37D3.v28.A2 RSSQSIVHSNGNTYFE HVR-L1 481 Hu37D3.v28.A4 RSSQSIVHSNGNTYLE HVR-L1 482 Hu37D3.v28.A6 RSSQSIVHSNGNTYLE HVR-L1 483 Hu37D3.v28.A8 RSSQSIVHSNGNTYFE HVR-L1 484 Hu37D3.v28.A10 RSSQSIVHSNGNTYFE HVR-L1 485 Hu37D3.v28.A12 RSSQSIVHSNGNTYLE HVR-L1 486 Hu37D3.v28.A14 RSSQSIVHSNGNTYLE HVR-L1 487 Hu37D3.v28.A16 RSSQSIVHSNGNTYFE HVR-L1 488 Hu37D3.v28.A18 RSSQSIVHSNGNTYFE HVR-L1 489 Hu37D3.v28.A20 RSSQSIVHSNGNTYLE HVR-L1 490 Hu37D3.v28.A22 RSSQSIVHSNGNTYLE HVR-L1 491 Hu37D3.v28.A24 RSSQSIVHSNGNTYFE HVR-L1 492 Hu37D3.v28.A26 RSSQSIVHSNGNTYFE HVR-L1 493 Hu37D3.v28.A28 RSSQSIVHSNGNTYLE HVR-L1 494 Hu37D3.v28.A30 RSSQSIVHSNGNTYLE HVR-L1 495 Hu37D3.v28.B1 RSSQSIVHSIGNTFFE HVR-L1 496 Hu37D3.v28.B2 RSSQSIVHSMGNTFFE HVR-L1 497 Hu37D3.v28.B3 RSSQSIVHSQGNTWFE HVR-L1 498 Hu37D3.v28.B4 RSSQSIVHSQGNTHFE HVR-L1 499 Hu37D3.v28.B6 RSSQSIVHSDGNTRFE HVR-L1 500 Hu37D3.v28.B7 RSSQSIVHSDGNTKFE HVR-L1 501 Hu37D3.v28.B8 RSSQSIVHSEGNTRFE HVR-L1 502 Hu37D3.v28.C1 RSSQSIVHSNNNTYFE HVR-L1 503 Hu37D3.v28.C2 RSSQSIVHSNDNTYFE HVR-L1 504 Hu37D3.v28.D1 RSSQSIVHANGNTYFE HVR-L1 505 Hu37D3.v28.E1 RSSQSIVHSNGNTYFE HVR-L1 506 Hu37D3.v28.E2 RSSQSIVQSNGNTYFE HVR-L1 507 Hu37D3.v28.E3 RSSQSIVDSDGNTYFE HVR-L1 508 Hu37D3.v28.F1 RSSQSIVHSNTNTYFE HVR-L1 509 Hu37D3.v28.F2 RSSQSIVHTNGNTYFE HVR-L1 510 Hu37D3.v28.F3 RSSQSIVHTNANTYFE HVR-L1 511 Hu37D3.v28.51 RSSQSIVHSHGNTYFE HVR-L1 512 Hu37D3.v28.52 RSSQSIVHSKGNTYFE HVR-L1 513 Hu37D3.v28.53 RSSQSIVHSRGNTYFE HVR-L1 514 Hu37D3.v28.54 RSSQSIVHSLGNTYFE HVR-L1 515 Hu37D3.v28.55 RSSQSIVHSNQNTYFE HVR-L1 516 Hu37D3.v28.56 RSSQSIVHSNYNTYFE HVR-L1 517 Hu37D3.v28.57 RSSQSIVHSNFNTYFE HVR-L1 518 Hu37D3.v29.1 RSSQSIVHSNGDTYFE HVR-L1 519 Hu37D3.v29.2 RSSQSIVHSNGQTYFE HVR-L1 520 Hu37D3.v29.3 RSSQSIVHSNGETYFE HVR-L1 521 Hu37D3.v29.4 RSSQSIVHSNGATYFE HVR-L1 522 Hu37D3.v29.5 RSSQSIVHSNGHTYFE HVR-L1 523 Hu37D3.v29.6 RSSQSIVHSNGETYFE HVR-L1 524 Hu37D3.v29.7 RSSQSIVHSNGLTYFE HVR-L1 525 Hu37D3.v29.8 RSSQSIVHSNADTYFE HVR-L1 526 Hu37D3.v29.9 RSSQSIVHSNAQTYFE HVR-L1 527 Hu37D3.v29.10 RSSQSIVHSNAETYFE HVR-L1 528 Hu37D3.v29.11 RSSQSIVHSNAATYFE HVR-L1 529 Hu37D3.v29.12 RSSQSIVHSNAHTYFE HVR-L1 530 Hu37D3.v29.13 RSSQSIVHSNAETYFE HVR-L1 531 Hu37D3.v29.14 RSSQSIVHSNALTYFE HVR-L1 532 Hu37D3-H9.v30.1 RSSQSIVHSGGNTYFE HVR-L1 533 Hu37D3-H9.v30.2 RSSQSIVHSTGNTYFE HVR-L1 534 Hu37D3-H9.v30.3 RSSQSIVHSVGNTYFE HVR-L1 535 Hu37D3-H9.v30.4 RSSQSIVHSLGNTYFE HVR-L1 536 Hu37D3-H9.v30.5 RSSQSIVHSIGNTYFE HVR-L1 537 Hu37D3-H9.v30.6 RSSQSIVHSPGNTYFE HVR-L1 538 Hu37D3-H9.v30.7 RSSQSIVHSFGNTYFE HVR-L1 539 Hu37D3-H9.v30.8 RSSQSIVHSYGNTYFE HVR-L1 540 Hu37D3-H9.v30.9 RSSQSIVHSHGNTYFE HVR-L1 541 Hu37D3-H9.v30.10 RSSQSIVHSFGNTYFE HVR-L1 542 Hu37D3-H9.v30.11 RSSQSIVHSRGNTYFE HVR-L1 543 Hu37D3-H9.v31.1 RSSQSIVHSNAGTYFE HVR-L1 544 Hu37D3-H9.v31.2 RSSQSIVHSNAVTYFE HVR-L1 545 Hu37D3-H9.v31.3 RSSQSIVHSNAITYFE HVR-L1 546 Hu37D3-H9.v31.4 RSSQSIVHSNAPTYFE HVR-L1 547 Hu37D3-H9.v31.5 RSSQSIVHSNAFTYFE HVR-L1 548 Hu37D3-H9.v31.6 RSSQSIVHSNAYTYFE HVR-L1 549 Hu37D3-H9.v31.7 RSSQSIVHSNARTYFE HVR-L1 550 Hu37D3-H9.v31.8 RSSQSIVHSNANVYFE HVR-L1 551 Hu37D3-H9.v31.9 RSSQSIVHSNANIYFE HVR-L1 552 Hu37D3-H9.v31.10 RSSQSIVHSNANPYFE HVR-L1 553 Hu37D3-H9.v31.11 RSSQSIVHSNANFYFE HVR-L1 554 Hu37D3-H9.v31.12 RSSQSIVHSNANYYFE HVR-L1 555 Hu37D3-H9.v31.13 RSSQSIVHSNANNYFE HVR-L1 556 Hu37D3-H9.v31.14 RSSQSIVHSNANRYFE HVR-L1 557 Human Tau 7-24 EFEVMEDHAGTYGLGDRK peptide 558 Human Tau 7-20 EFEVMEDHAGTYGL peptide 560 Hu37D3.v39 heavy EVQLVESGGG LVQPGGSLRL SCAASGLIFR SYGMSWVRQA chain variable PGKGLEWVAT INSGGTYTYY PDSVKGRFTI SRDNSKNTLY region (VH) LQMNSLRAED TAVYYCANSY SGAMDYWGQG TLVTVSS 561 Hu37D3.v39 light EDQLTQSPSS LSASVGDRVT ITCRSSQSIV HSNGNTYLEW chain variable YQQKPGKSPK LLIYKVSNRF SGVPSRFSGS GSGTDFTLTI region (VL) SSLQPEDFAT YYCFQGSLVP WTFGQGTKVE IK 562 Hu37D3.v39 HVR- SYGMS H1 563 Hu37D3.v39 HVR- TINSGGTYTYYPDSVKG H2 564 Hu37D3.v39 HVR- SYSGAMDY H3 565 Hu37D3.v39 HVR- RSSQSIVHSNGNTYLE L1 566 Hu37D3.v39 HVR- KVSNRFS L2 567 Hu37D3.v39 HVR- FQGSLVPWT L3 568 Hu37D3.v39 IgG4- EVQLVESGGG LVQPGGSLRL SCAASGLIFR SYGMSWVRQA S228P.YTE heavy PGKGLEWVAT INSGGTYTYY PDSVKGRFTI SRDNSKNTLY chain LQMNSLRAED TAVYYCANSY SGAMDYWGQG TLVTVSSAST KGPSVFPLAP CSRSTSESTA ALGCLVKDYF PEPVTVSWNS GALTSGVHTF PAVLQSSGLY SLSSVVTVPS SSLGTKTYTC NVDHKPSNTK VDKRVESKYG PPCPPCPAPE FLGGPSVFLF PPKPKDTLYI TREPEVTCVV VDVSQEDPEV QFNWYVDGVE VHNAKTKPRE EQFNSTYRVV SVLTVLHQDW LNGKEYKCKV SNKGLPSSIE KTISKAKGQP REPQVYTLPP SQEEMTKNQV SLTCLVKGFY PSDIAVEWES NGQPENNYKT TPPVLDSDGS FFLYSRLTVD KSRWQEGNVF SCSVMHEALH NHYTQKSLSL SLGK 569 Hu37D3.v39 IgG4- EDQLTQSPSS LSASVGDRVT ITCRSSQSIV HSNGNTYLEW S228P.YTE light YQQKPGKSPK LLIYKVSNRF SGVPSRFSGS GSGTDFTLTI chain SSLQPEDFAT YYCFQGSLVP WTFGQGTKVE IKRTVAAPSV FIFPPSDEQL KSGTASVVCL LNNFYPREAK VQWKVDNALQ SGNSQESVTE QDSKDSTYSL SSTLTLSKAD YEKHKVYACE VTHQGLSSPV TKSFNRGEC 570 Hu37D3.v40 heavy EVQLVESGGG LVQPGGSLRL SCAASGLIFR SYGMSWVRQA chain variable PGKGLEWVAT INSGGTYTYY PDSVKGRFTI SRDNSKNTLY region (VH) LQMNSLRAED TAVYYCANSY SGAMDYWGQG TLVTVSS 571 Hu37D3.v40 light EDQLTQSPSS LSASVGDRVT ITCRSSQSIV HSNTNTYFEW chain variable YQQKPGKSPK LLIYKVSNRF SGVPSRFSGS GSGTDFTLTI region (VL) SSLQPEDFAT YYCFQGSLVP WTFGQGTKVE IK 572 Hu37D3.v40 HVR- SYGMS H1 573 Hu37D3.v40 HVR- TINSGGTYTYYPDSVKG H2 574 Hu37D3.v40 HVR- SYSGAMDY H3 575 Hu37D3.v40 HVR- RSSQSIVHSNTNTYFE L1 576 Hu37D3.v40 HVR- KVSNRFS L2 577 Hu37D3.v40 HVR- FQGSLVPWT L3 578 Hu37D3.v40 IgG4- EVQLVESGGG LVQPGGSLRL SCAASGLIFR SYGMSWVRQA S228P.YTE heavy PGKGLEWVAT INSGGTYTYY PDSVKGRFTI SRDNSKNTLY chain LQMNSLRAED TAVYYCANSY SGAMDYWGQG TLVTVSSAST KGPSVFPLAP CSRSTSESTA ALGCLVKDYF PEPVTVSWNS GALTSGVHTF PAVLQSSGLY SLSSVVTVPS SSLGTKTYTC NVDHKPSNTK VDKRVESKYG PPCPPCPAPE FLGGPSVFLF PPKPKDTLYI TREPEVTCVV VDVSQEDPEV QFNWYVDGVE VHNAKTKPRE EQFNSTYRVV SVLTVLHQDW LNGKEYKCKV SNKGLPSSIE KTISKAKGQP REPQVYTLPP SQEEMTKNQV SLTCLVKGFY PSDIAVEWES NGQPENNYKT TPPVLDSDGS FFLYSRLTVD KSRWQEGNVF SCSVMHEALH NHYTQKSLSL SLGK 579 Hu37D3.v40 IgG4- EDQLTQSPSS LSASVGDRVT ITCRSSQSIV HSNTNTYFEW S228P.YTE light YQQKPGKSPK LLIYKVSNRF SGVPSRFSGS GSGTDFTLTI chain SSLQPEDFAT YYCFQGSLVP WTFGQGTKVE IKRTVAAPSV FIFPPSDEQL KSGTASVVCL LNNFYPREAK VQWKVDNALQ SGNSQESVTE QDSKDSTYSL SSTLTLSKAD YEKHKVYACE VTHQGLSSPV TKSFNRGEC 580 Hu37D3.v41 heavy EVQLVESGGG LVQPGGSLRL SCAASGLIFR SYGMSWVRQA chain variable PGKGLEWVAT INSGGTYTYY PDSVKGRFTI SRDNSKNTLY region (VH) LQMNSLRAED TAVYYCANSY SGAMDYWGQG TLVTVSS 581 Hu37D3.v41 light EDQLTQSPSS LSASVGDRVT ITCRSSQSIV HSNGQTYFEW chain variable YQQKPGKSPK LLIYKVSNRF SGVPSRFSGS GSGTDFTLTI region (VL) SSLQPEDFAT YYCFQGSLVP WTFGQGTKVE IK 582 Hu37D3.v41 HVR- SYGMS H1 583 Hu37D3.v41 HVR- TINSGGTYTYYPDSVKG H2 584 Hu37D3.v41 HVR- SYSGAMDY H3 585 Hu37D3.v41 HVR- RSSQSIVHSNGQTYFE L1 586 Hu37D3.v41 HVR- KVSNRFS L2 587 Hu37D3.v41 HVR- FQGSLVPWT L3 588 Hu37D3.v41 IgG4- EVQLVESGGG LVQPGGSLRL SCAASGLIFR SYGMSWVRQA S228P.YTE heavy PGKGLEWVAT INSGGTYTYY PDSVKGRFTI SRDNSKNTLY chain LQMNSLRAED TAVYYCANSY SGAMDYWGQG TLVTVSSAST KGPSVFPLAP CSRSTSESTA ALGCLVKDYF PEPVTVSWNS GALTSGVHTF PAVLQSSGLY SLSSVVTVPS SSLGTKTYTC NVDHKPSNTK VDKRVESKYG PPCPPCPAPE FLGGPSVFLF PPKPKDTLYI TREPEVTCVV VDVSQEDPEV QFNWYVDGVE VHNAKTKPRE EQFNSTYRVV SVLTVLHQDW LNGKEYKCKV SNKGLPSSIE KTISKAKGQP REPQVYTLPP SQEEMTKNQV SLTCLVKGFY PSDIAVEWES NGQPENNYKT TPPVLDSDGS FFLYSRLTVD KSRWQEGNVF SCSVMHEALH NHYTQKSLSL SLGK 589 Hu37D3.v41 IgG4- EDQLTQSPSS LSASVGDRVT ITCRSSQSIV HSNGQTYFEW S228P.YTE light YQQKPGKSPK LLIYKVSNRF SGVPSRFSGS GSGTDFTLTI chain SSLQPEDFAT YYCFQGSLVP WTFGQGTKVE IKRTVAAPSV FIFPPSDEQL KSGTASVVCL LNNFYPREAK VQWKVDNALQ SGNSQESVTE QDSKDSTYSL SSTLTLSKAD YEKHKVYACE VTHQGLSSPV TKSFNRGEC 590 Hu37D3-H9.v1 EVQLVESGGG LVQPGGSLRL SCAASGLIFR SYGMSWVRQA IgG4-S228P heavy PGKGLEWVAT INSGGTYTYY PDSVKGRFTI SRDNSKNTLY chain LQMNSLRAED TAVYYCANSY SGAMDYWGQG TLVTVSSAST KGPSVFPLAP CSRSTSESTA ALGCLVKDYF PEPVTVSWNS GALTSGVHTF PAVLQSSGLY SLSSVVTVPS SSLGTKTYTC NVDHKPSNTK VDKRVESKYG PPCPPCPAPE FLGGPSVFLF PPKPKDTLMI SRTPEVTCVV VDVSQEDPEV QFNWYVDGVE VHNAKTKPRE EQFNSTYRVV SVLTVLHQDW LNGKEYKCKV SNKGLPSSIE KTISKAKGQP REPQVYTLPP SQEEMTKNQV SLTCLVKGFY PSDIAVEWES NGQPENNYKT TPPVLDSDGS FFLYSRLTVD KSRWQEGNVF SCSVMHEALH NHYTQKSLSL SLGK 591 Hu37D3-H9.v1 EDQLTQSPSS LSASVGDRVT ITCRSSQSIV HSNGNTYFEW IgG4 light chain YQQKPGKSPK LLIYKVSNRF SGVPSRFSGS GSGTDFTLTI SSLQPEDFAT YYCFQGSLVP WTFGQGTKVE IKRTVAAPSV FIFPPSDEQL KSGTASVVCL LNNFYPREAK VQWKVDNALQ SGNSQESVTE QDSKDSTYSL SSTLTLSKAD YEKHKVYACE VTHQGLSSPV TKSFNRGEC 592 MAPT(10-24) VMEDHAGTYGLGDRK 593 MAPT(2-24) AEPRQEFEVMEDHAGTYGLGDRK 594 MAPT(2-34) AEPRQEFEVMEDHAGTYGLGDRKDQGGYTMHQD 595 MAPT(10-44) VMEDHAGTYGLGDRKDQGGYTMHQDQEGDTDAGLK 596 MAPT(2-24)Y18A AEPRQEFEVMEDHAGTAGLGDRK 597 MAPT(2-24)L20A AEPRQEFEVMEDHAGTYGAGDRK 598 hu113F5-F7.LC1 DIQMTQSPSS LSASVGDRVT ITCKASQNVG TAVAWYQQKP GKSPKLLIYS ASRRFSGVPS RFSGSGSGTD FTLTISSLQP EDFATYFCQQ FSTYPYTFGQ GTKVEIK 599 hu113F5-F7.LC2 DIQMTQSPSS LSASVGDRVT ITCKASQNVG TAVAWYQQKP GKAPKLLIYS ASRRFSGVPS RFSGSGSGTD FTLTISSLQP EDFATYFCQQ FSTYPYTFGQ GTKVEIK 600 hu113F5-F7.LC3 DIQMTQSPSS LSASVGDRVT ITCKASQNVG TAVAWYQQKP GKSPKLLIYS ASRRFSGVPS RFSGSGSGTD FTLTISSLQP EDFATYYCQQ FSTYPYTFGQ GTKVEIK 601 hu113F5-F7.LC4 DIQMTQSPSS LSASVGDRVT ITCKASQNVG TAVAWYQQKP GKAPKLLIYS ASRRFSGVPS RFSGSGSGTD FTLTISSLQP EDFATYYCQQ FSTYPYTFGQ GTKVEIK 603 Hu37D3-H9.v76 EVQLVESGGG LVQPGGSLRL SCAASGLIFR SYGMSWVRQA heavy chain variable PGKGLEWVAT INSGGTRTYY PDSVKGRFTI SRDNSKNTLY region (VH) LQMNSLRAED TAVYYCANSY SGAMDYWGQG TLVTVSS 604 Hu37D3-H9.v76 DDLLTQTPLS LPVTPGQPAS ISCRSSQSIV HSNGNTYLEW light chain variable YLQKPGQSPQ LLIYKVSNRF SGVPDRFSGS GSGTDFTLKI region (VL) SRVEAEDVGV YYCFQGTLVP WTFGQGTKVE IK 605 Hu37D3-H9.v76 SYGMS HVR-H1 Hu37D3-H9.v83 HVR-H1 Hu37D3-H9.v93 HVR-H1 606 Hu37D3-H9.v76 TINSGGTRTYYPDSVKG HVR-H2 Hu37D3-H9.v83 HVR-H2 Hu37D3-H9.v93 HVR-H2 607 Hu37D3-H9.v76 SYSGAMDY HVR-H3 Hu37D3-H9.v83 HVR-H3 Hu37D3-H9.v93 HVR-H3 608 Hu37D3-H9.v76 RSSQSIVHSNGNTYLE HVR-L1 Hu37D3-H9.v83 HVR-L1 Hu37D3-H9.v93 HVR-L1 609 Hu37D3-H9.v76 KVSNRFS HVR-L2 Hu37D3-H9.v83 HVR-L2 Hu37D3-H9.v93 HVR-L2 610 Hu37D3-H9.v76 FQGTLVPWT HVR-L3 Hu37D3-H9.v83 HVR-L3 Hu37D3-H9.v93 HVR-L3 611 Hu37D3-H9.v76 EVQLVESGGG LVQPGGSLRL SCAASGLIFR SYGMSWVRQA IgG4-S228P.YTE PGKGLEWVAT INSGGTRTYY PDSVKGRFTI SRDNSKNTLY heavy chain LQMNSLRAED TAVYYCANSY SGAMDYWGQG TLVTVSSAST KGPSVFPLAP CSRSTSESTA ALGCLVKDYF PEPVTVSWNS GALTSGVHTF PAVLQSSGLY SLSSVVTVPS SSLGTKTYTC NVDHKPSNTK VDKRVESKYG PPCPPCPAPE FLGGPSVFLF PPKPKDTLYI TREPEVTCVV VDVSQEDPEV QFNWYVDGVE VHNAKTKPRE EQFNSTYRVV SVLTVLHQDW LNGKEYKCKV SNKGLPSSIE KTISKAKGQP REPQVYTLPP SQEEMTKNQV SLTCLVKGFY PSDIAVEWES NGQPENNYKT TPPVLDSDGS FFLYSRLTVD KSRWQEGNVF SCSVMHEALH NHYTQKSLSL SLGK 612 Hu37D3-H9.v76 EVQLVESGGG LVQPGGSLRL SCAASGLIFR SYGMSWVRQA IgG4-S228P.YTE PGKGLEWVAT INSGGTRTYY PDSVKGRFTI SRDNSKNTLY des-K heavy chain LQMNSLRAED TAVYYCANSY SGAMDYWGQG TLVTVSSAST KGPSVFPLAP CSRSTSESTA ALGCLVKDYF PEPVTVSWNS GALTSGVHTF PAVLQSSGLY SLSSVVTVPS SSLGTKTYTC NVDHKPSNTK VDKRVESKYG PPCPPCPAPE FLGGPSVFLF PPKPKDTLYI TREPEVTCVV VDVSQEDPEV QFNWYVDGVE VHNAKTKPRE EQFNSTYRVV SVLTVLHQDW LNGKEYKCKV SNKGLPSSIE KTISKAKGQP REPQVYTLPP SQEEMTKNQV SLTCLVKGFY PSDIAVEWES NGQPENNYKT TPPVLDSDGS FFLYSRLTVD KSRWQEGNVF SCSVMHEALH NHYTQKSLSL SLG 613 Hu37D3-H9.v76 DDLLTQTPLS LPVTPGQPAS ISCRSSQSIV HSNGNTYLEW IgG4-S228P.YTE YLQKPGQSPQ LLIYKVSNRF SGVPDRFSGS GSGTDFTLKI light chain SRVEAEDVGV YYCFQGTLVP WTFGQGTKVE IKRTVAAPSV FIFPPSDEQL KSGTASVVCL LNNFYPREAK VQWKVDNALQ SGNSQESVTE QDSKDSTYSL SSTLTLSKAD YEKHKVYACE VTHQGLSSPV TKSFNRGEC 614 Hu37D3-H9.v83 EVQLLESGGG LVQPGGSLRL SCAASGLIFR SYGMSWVRQA heavy chain variable PGKGLEWVAT INSGGTRTYY PDSVKGRFTI SRDNSKNTLY region (VH) LQMNSLRAED TAVYYCANSY SGAMDYWGQG TLVTVSS 615 Hu37D3-H9.v83 DDLLTQSPLS LPVTLGQPAS ISCRSSQSIV HSNGNTYLEW light chain variable YQQRPGQSPR LLIYKVSNRF SGVPDRFSGS GSGTDFTLKI region (VL) SRVEAEDVGV YYCFQGTLVP WTFGQGTKVE IK 616 Hu37D3-H9.v83 EVQLLESGGG LVQPGGSLRL SCAASGLIFR SYGMSWVRQA IgG4-S228P.YTE PGKGLEWVAT INSGGTRTYY PDSVKGRFTI SRDNSKNTLY heavy chain LQMNSLRAED TAVYYCANSY SGAMDYWGQG TLVTVSSAST KGPSVFPLAP CSRSTSESTA ALGCLVKDYF PEPVTVSWNS GALTSGVHTF PAVLQSSGLY SLSSVVTVPS SSLGTKTYTC NVDHKPSNTK VDKRVESKYG PPCPPCPAPE FLGGPSVFLF PPKPKDTLYI TREPEVTCVV VDVSQEDPEV QFNWYVDGVE VHNAKTKPRE EQFNSTYRVV SVLTVLHQDW LNGKEYKCKV SNKGLPSSIE KTISKAKGQP REPQVYTLPP SQEEMTKNQV SLTCLVKGFY PSDIAVEWES NGQPENNYKT TPPVLDSDGS FFLYSRLTVD KSRWQEGNVF SCSVMHEALH NHYTQKSLSL SLGK 617 Hu37D3-H9.v83 EVQLLESGGG LVQPGGSLRL SCAASGLIFR SYGMSWVRQA IgG4-S228PYTE PGKGLEWVAT INSGGTRTYY PDSVKGRFTI SRDNSKNTLY des-K heavy chain LQMNSLRAED TAVYYCANSY SGAMDYWGQG TLVTVSSAST KGPSVFPLAP CSRSTSESTA ALGCLVKDYF PEPVTVSWNS GALTSGVHTF PAVLQSSGLY SLSSVVTVPS SSLGTKTYTC NVDHKPSNTK VDKRVESKYG PPCPPCPAPE FLGGPSVFLF PPKPKDTLYI TREPEVTCVV VDVSQEDPEV QFNWYVDGVE VHNAKTKPRE EQFNSTYRVV SVLTVLHQDW LNGKEYKCKV SNKGLPSSIE KTISKAKGQP REPQVYTLPP SQEEMTKNQV SLTCLVKGFY PSDIAVEWES NGQPENNYKT TPPVLDSDGS FFLYSRLTVD KSRWQEGNVF SCSVMHEALH NHYTQKSLSL SLG 618 Hu37D3-H9.v83 DDLLTQSPLS LPVTLGQPAS ISCRSSQSIV HSNGNTYLEW IgG4-S228PYTE YQQRPGQSPR LLIYKVSNRF SGVPDRFSGS GSGTDFTLKI light chain SRVEAEDVGV YYCFQGTLVP WTFGQGTKVE IKRTVAAPSV FIFPPSDEQL KSGTASVVCL LNNFYPREAK VQWKVDNALQ SGNSQESVTE QDSKDSTYSL SSTLTLSKAD YEKHKVYACE VTHQGLSSPV TKSFNRGEC 619 Hu37D3-H9.v93 EVQLVESGGG LVQPGGSLRL SCAASGLIFR SYGMSWVRQA heavy chain variable PGKGLEWVAT INSGGTRTYY PDSVKGRFTI SRDNSKNTLY region (VH) LQMNSLRAED TAVYYCANSY SGAMDYWGQG TLVTVSS 620 Hu37D3-H9.v93 EDLLTQSPSS LSASVGDRVT ITCRSSQSIV HSNGNTYLEW light chain variable YQQKPGKSPK LLIYKVSNRF SGVPSRFSGS GSGTDFTLTI region (VL) SSLQPEDFAT YYCFQGTLVP WTFGQGTKVE IK 621 Hu37D3-H9.v93 EVQLVESGGG LVQPGGSLRL SCAASGLIFR SYGMSWVRQA IgG4-S228P.YTE PGKGLEWVAT INSGGTRTYY PDSVKGRFTI SRDNSKNTLY heavy chain LQMNSLRAED TAVYYCANSY SGAMDYWGQG TLVTVSSAST KGPSVFPLAP CSRSTSESTA ALGCLVKDYF PEPVTVSWNS GALTSGVHTF PAVLQSSGLY SLSSVVTVPS SSLGTKTYTC NVDHKPSNTK VDKRVESKYG PPCPPCPAPE FLGGPSVFLF PPKPKDTLYI TREPEVTCVV VDVSQEDPEV QFNWYVDGVE VHNAKTKPRE EQFNSTYRVV SVLTVLHQDW LNGKEYKCKV SNKGLPSSIE KTISKAKGQP REPQVYTLPP SQEEMTKNQV SLTCLVKGFY PSDIAVEWES NGQPENNYKT TPPVLDSDGS FFLYSRLTVD KSRWQEGNVF SCSVMHEALH NHYTQKSLSL SLGK 622 Hu37D3-H9.v93 EVQLVESGGG LVQPGGSLRL SCAASGLIFR SYGMSWVRQA IgG4-S228P.YTE PGKGLEWVAT INSGGTRTYY PDSVKGRFTI SRDNSKNTLY des-K heavy chain LQMNSLRAED TAVYYCANSY SGAMDYWGQG TLVTVSSAST KGPSVFPLAP CSRSTSESTA ALGCLVKDYF PEPVTVSWNS GALTSGVHTF PAVLQSSGLY SLSSVVTVPS SSLGTKTYTC NVDHKPSNTK VDKRVESKYG PPCPPCPAPE FLGGPSVFLF PPKPKDTLYI TREPEVTCVV VDVSQEDPEV QFNWYVDGVE VHNAKTKPRE EQFNSTYRVV SVLTVLHQDW LNGKEYKCKV SNKGLPSSIE KTISKAKGQP REPQVYTLPP SQEEMTKNQV SLTCLVKGFY PSDIAVEWES NGQPENNYKT TPPVLDSDGS FFLYSRLTVD KSRWQEGNVF SCSVMHEALH NHYTQKSLSL SLG 623 Hu37D3-H9.v93 EDLLTQSPSS LSASVGDRVT ITCRSSQSIV HSNGNTYLEW IgG4-S228P.YTE YQQKPGKSPK LLIYKVSNRF SGVPSRFSGS GSGTDFTLTI light chain SSLQPEDFAT YYCFQGTLVP WTFGQGTKVE IKRTVAAPSV FIFPPSDEQL KSGTASVVCL LNNFYPREAK VQWKVDNALQ SGNSQESVTE QDSKDSTYSL SSTLTLSKAD YEKHKVYACE VTHQGLSSPV TKSFNRGEC 624 residues 2 to 24 of AEPRQEFEVMEDHAGTYGLGDRK human Tau 

1. (canceled)
 2. (canceled)
 3. (canceled)
 4. An isolated antibody that binds to human Tau, wherein the antibody comprises: a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 342; HVR-H2 comprising the amino acid sequence of SEQ ID NO: 343; HVR-H3 comprising the amino acid sequence of SEQ ID NO: 344; HVR-L1 comprising the amino acid sequence of SEQ ID NO: 608; HVR-L2 comprising the amino acid sequence of SEQ ID NO: 609; and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 610; or b) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 605; HVR-H2 comprising the amino acid sequence of SEQ ID NO: 606; HVR-H3 comprising the amino acid sequence of SEQ ID NO: 607; HVR-L1 comprising the amino acid sequence of SEQ ID NO: 345; HVR-L2 comprising the amino acid sequence of SEQ ID NO: 346; and HVR-L3 comprising the amino acid sequence of SEQ ID NO:
 347. 5. The isolated antibody of claim 4, wherein the antibody binds to monomeric Tau, oligomeric Tau, non-phosphorylated Tau, and phosphorylated Tau.
 6. The isolated antibody of claim 4, wherein the antibody binds an epitope within amino acids 2 to 24 of mature human Tau.
 7. The isolated antibody of claim 4, which is a monoclonal antibody.
 8. The isolated antibody of claim 4, which is a human, humanized, or chimeric antibody.
 9. The antibody of claim 4, which is an antibody fragment that binds human Tau.
 10. The antibody of claim 4, wherein the human Tau comprises the sequence of SEQ ID NO:
 2. 11. (canceled)
 12. (canceled)
 13. (canceled)
 14. The isolated antibody of claim 4, wherein the antibody comprises a heavy chain variable region comprising the sequence of SEQ ID NO: 340; and a light chain variable region comprising a sequence selected from SEQ ID NOs: 608, 609, and
 610. 15. The isolated antibody of claim 4, wherein the antibody comprises a heavy chain variable region comprising a sequence selected from SEQ ID NOs: 605, 606, and 607; and a light chain variable region comprising the sequence of SEQ ID NO:
 341. 16. (canceled)
 17. (canceled)
 18. (canceled)
 19. (canceled)
 20. (canceled)
 21. (canceled)
 22. The isolated antibody of claim 4, wherein the antibody is an IgG₁ or an IgG₄ antibody.
 23. The isolated antibody of claim 22, wherein the antibody is an IgG₄ antibody.
 24. The isolated antibody of claim 23, wherein the antibody comprises M252Y, S254T, and T256E mutations.
 25. The isolated antibody of claim 24, wherein the antibody comprises an S228P mutation.
 26. (canceled)
 27. The isolated antibody of claim 4, wherein the antibody binds monomeric Tau with a K_(D) of less than 5 nM.
 28. The isolated antibody of claim 4, wherein the antibody binds to human monomeric Tau with a K_(D) of less than 1 nM.
 29. The isolated antibody of claim 27, wherein K_(D) is determined by surface plasmon resonance.
 30. The isolated antibody of claim 4, which binds cynomolgus monkey Tau.
 31. An isolated nucleic acid that encodes the antibody of claim
 4. 32. A host cell comprising the nucleic acid of claim
 31. 33. A method of producing an antibody comprising culturing the host cell of claim 32 under conditions suitable for producing the antibody.
 34. An immunoconjugate comprising the isolated antibody of claim 4 and a second therapeutic agent.
 35. A labeled antibody comprising the antibody of claim 4 and a detectable label.
 36. A pharmaceutical composition comprising the isolated antibody of claim 4 and a pharmaceutically acceptable carrier.
 37. A method of treating a Tau protein associated disease comprising administering to an individual with a Tau protein related disease the antibody of claim
 4. 38. The method of claim 37, wherein the Tau protein associated disease is a tauopathy.
 39. The method of claim 38, wherein the tauopathy is a neurodegenerative tauopathy.
 40. The method of claim 37, wherein the tauopathy is selected from Alzheimer's Disease, amyotrophic lateral sclerosis, Parkinson's disease, Creutzfeldt-Jacob disease, Dementia pugilistica, Down Syndrome, Gerstmann-Straussler-Scheinker disease, inclusion-body myositis, prion protein cerebral amyloid angiopathy, traumatic brain injury, amyotrophic lateral sclerosis/parkinsonism-dementia complex of Guam, Non-Guamanian motor neuron disease with neurofibrillary tangles, argyrophilic grain dementia, corticobasal sdegeneration, diffuse neurofibrillary tangles with calcification, frontotemporal dementia, frontotemporal dementia with parkinsonism linked to chromosome 17, Hallervorden-Spatz disease, multiple system atrophy, Niemann-Pick disease type C, Pallido-ponto-nigral degeneration, Pick's disease, progressive subcortical gliosis, progressive supranuclear palsy, Subacute sclerosing panencephalitis, Tangle only dementia, Postencephalitic Parkinsonism, and Myotonic dystrophy.
 41. The method of claim 38, wherein the tauopathy is Alzheimer's disease or progressive supranuclear palsy.
 42. A method of retaining or increasing cognitive memory capacity or slowing memory loss in an individual, comprising administering the antibody of claim
 4. 43. A method of reducing the level of Tau protein, non-phosphorylated Tau protein, phosphorylated Tau protein, or hyperphosphorylated Tau protein in an individual with a tauopathy, comprising administering the antibody of claim
 4. 44. The method of claim 37, wherein the method comprises administering at least one additional therapy.
 45. The method of claim 44, wherein the additional therapy is selected from neurological drugs, corticosteroids, antibiotics, antiviral agents, anti-Tau antibodies, Tau inhibitors, anti-amyloid beta antibodies, beta-amyloid aggregation inhibitors, anti-BACE1 antibodies, BACE1 inhibitors, cholinesterase inhibitors (ChEIs), NMDA receptor antagonists, and nutritional supplements. 46.-62. (canceled)
 63. A method of detecting neurofibrillary tangles, neuropil threads, or dystrophic neuritis comprising contacting a sample with the antibody of claim
 4. 64. The method of claim 63, wherein the sample is a brain sample, a cerebrospinal fluid sample, or a blood sample.
 65. The method of claim 45, wherein the ChEI is tacrine, rivastigmine, donepezil, or galantamine; and wherein the NMDA receptor antagonist is memantine.
 66. The method of claim 45, wherein the anti-amyloid beta antibody is crenezumab. 